Method of delivering material or stimulus to a biological subject

ABSTRACT

A device for delivery of material or stimulus to targets within a body to produce a desired response, the targets being at least one of cells of interest, cell organelles of interest and cell nuclei of interest. The device includes a number of projections for penetrating a body surface, with the number of projections being selected to produce a desired response, and the number being at least 500. A spacing between projections is also at least partially determined based on an arrangement of the targets within the body.

CROSS-REFERENCE TO RELATED APPLICATION(S)

The current application is a continuation of U.S. Ser. No. 11/496,053filed on Jul. 27, 2006, which is a continuation-in-part (CIP) ofInternational Application Number PCT/GB2005/000336 which designates theU.S. and has an International Filing Date of 31 Jan. 2005 and which waspublished as International Publication No. WO2005/072630 on 11 Aug.2005. The entirety of International Application Number PCT/GB2005/000336is hereby incorporated herein by reference.

BACKGROUND Technical Field

The present invention relates to a device for delivery of material orstimulus to targets within a body to produce a desired response, and inparticular to a device including a number of projections for penetratinga body surface. The invention can also relate to devices for deliveringbioactive substances and other stimuli to living cells, to methods ofmanufacture of the device and to various uses of the device.

Description of the Related Art

The reference in this specification to any prior publication (orinformation derived from it), or to any matter which is known, is not,and should not be taken as an acknowledgment or admission or any form ofsuggestion that the prior publication (or information derived from it)or known matter forms part of the common general knowledge in the fieldof endeavor to which this specification relates.

In recent years, attempts have been made to devise new methods ofdelivering drugs and other bioactive materials, for vaccination andother purposes, which provide alternatives that are more convenientand/or enhanced in performance to the customary routes of administrationsuch as intramuscular and intradermal injection. Limitations ofintradermal injection include: cross-contamination through needle-stickinjuries in health workers; injection phobia from a needle and syringe;and most importantly, as a result of its comparatively large scale andmethod of administration, the needle and syringe cannot target key cellsin the outer skin layers (FIG. 11 (a)). This is a serious limitation tomany existing and emerging strategies for the prevention, treatment andmonitoring of a range of untreatable diseases.

The skin structure is shown in FIG. 11, with a summary of key existingdelivery methods. Non-invasive methods of delivery through the skin havebeen used, including patches, liquid solutions and creams. Their successis dependent upon the ability to breach the semi-permeable stratumcorneum (SC) into the viable epidermis. Typically, larger biomoleculesare unable to breach this barrier.

Alternatively, there are many more “invasive” means to breach the SC forpharmaceutical delivery to the viable epidermis. Simple methods include:tape stripping with an abrasive tape to or sandpaper and the applicationof depilatory agents. Amongst the more advanced technologies areelectroporation, ablation by laser or heat, radiofrequency high voltagecurrents, iontopheresis, liposomes, sonophoresis. Many of theseapproaches remain untested for complex entities such as vaccines andimmunotherapies. Moreover, they do not specifically deliver entitieswithin key skin cells.

Needle-free injection approaches include the high-speed liquid jetinjector, which had a rise and fall in popularity in the mid twentiethcentury—and has recently seen a resurgence (Furth, P. A., Shamay, A. &Henninghausen, L. (1995) Gene transfer into mammalian cells by jetinjection. Hybridoma, 14:149-152.). However, this method delivers jetsof liquid to the epidermis and dermis (labelled (c) in Fig A), usuallywith a diameter >100 μm and not within key cells. Furthermore, as aresult of the concentrated jet momentum, many skin cells die. Deliveryinto the dermis also leads to patients reporting pain from injection.

The ballistic, needle-free delivery of microparticles (or gene gun)offers a route for delivering biological agents directly into cells ofthe skin. In this needle-free technique, pharmaceutical orimmunomodulatory agents, formulated as or coated to particles, areaccelerated in a high-speed gas jet at sufficient momentum to penetratethe skin (or mucosal) layer and to achieve a pharmacological effect. Aschematic of microparticles in the skin following ballistic delivery isshown in FIG. 11(b). The ability of this “scatter gun” approach todeliver genes and drugs to epidermal cells is highly limited andsensitive to biological variability in skin properties on the dynamichigh strain rate ballistics process. These effects are discussed inKendall, M. A. F., Rishworth, S., Carter, F. V. & Mitchell, T. J. (2004)“The effects of relative humidity and ambient temperature on theballistic delivery of micro-particles into excised porcine skin.” J.Investigative Dermatology, 122(3):739-746.); and Kendall, M. A. F.,Mitchell, T. J. & Wrighton-Smith, P. (2004) Intradermal ballisticdelivery of microparticles into excised human skin for drug and vaccineapplications. J. Biomechanics, 37(11):1733-1741.

First, the ballistic delivery of particles into the skin to targetepidermal cells is extremely sensitive to the small variations in thestratum corneum-including the stratum corneum thickness, which variesmassively with body site, age, sex, race and exposure to climaticconditions. (The quasi-static loading of skin with micro-nanostructureswould be less sensitive to these differences).

Second, it has been shown that even when all these parameters arestrictly controlled—and the only parameter varied is the climaticrelative humidity (15%-95%), or, independently, temperature (20° C.−40°C.)—the result is a large variation in penetration depth. These resultsare shown in FIG. 12, with particle penetration as a function of ambientrelative humidity (FIG. 12A) and ambient temperature (FIG. 12B) plottedalong with theoretical calculations of particle penetration and measuredstratum corneum thickness. This variation alone is significant andsufficient to make the difference between particles breaching thestratum corneum, or not.

The compound effect of these two (and other) sources of variability isthe gene gun/biolistics process does not consistently target epidermalcells-leading to inconsistent biological responses (e.g., in DNAvaccination).

Interestingly, it has also been shown that the high strain-rate loadingof the skin under ballistic particle impact (approximately 10⁶ persecond) increases the stratum corneum breaking stress by up to a factorof 10 compared to quasi-static values-due to a ductile-to-brittle changein the skin mechanical properties. This means that the tissue is moredifficult to penetrate as the particle impact velocity is increased.Therefore it is desirable to devise a way to delivermicro/nanostructures to the skin at lower strain-rates than theballistic approach to exploit the weaker stratum corneum.

When the microparticles are delivered to the skin, it is unclear whetherthere are any adverse longer term effects. For example, in the case ofinsoluble particles, many of them slough off with the usual skinturnover. However, gold particles have been detected in the lymph nodesfollowing ballistic particle delivery-presumably by migration withLangerhans cells. Uncertainty of adverse effects of these deliveredmaterials would be removed by delivery routes that do not leave suchmaterials in tissue site.

Moreover, when the microparticles successfully target cells, there is asignificant probability they kill the cells they target. Consider atypical ballistic delivery condition: over 1 million 2-3 um diametergold particles coated in DNA to the skin at 400-600 m/s, over a targetdiameter of 4 mm (Kendall, M. A. F., Mulholland, W. J Tirlapur, U. K.,Arbuthnott, E. S. & Armitage, M. (2003) Targeted delivery ofmicro-particles to epithelial cells for immunotherapy and vaccines: anexperimental and probabilistic study. 6th International Conference onCellular Engineering. Aug. 20-22, 2003, Sydney, Australia.). Reportedexperiments with these conditions using cell death stains (ethidiumbromide/acridin orange) show that microparticles impacting the skin dokill cells (McSloy, N. J., Raju, P. A. & Kendall, M. A. F. (2004) Theeffects of shock waves and particle penetration in skin on cellviability following gene gun delivery. British Society for Gene Therapy,1st Annual Conference. Oxford, UK, Mar. 28-30, 2004; Raju, P. A. &Kendall, M. A. F. (2004) Epidermal cell viability following theballistic delivery of DNA vaccine microparticles. DNA Vaccines 2004-theGene Vaccine Conference. 17-19 Nov. 2004, Monte Carlo, Monaco.).

FIG. 13A shows the percentage of cells that had membrane rupture (i.e.,death) as a function of the localized particle channel density. In FIG.13B we see schematically the way the data in FIG. 13A was achieved,relating “tracks” left by particle penetration to the death of cells ina layer of the viable epidermis. Clearly, FIG. 13A shows that at achannel density above 0.01 channels/micron, all the cells in that layerare dead. Indeed, FIG. 13C shows that cells are killed when the particleis passing up to 10 μm outside of the cell boundary. The mechanism ofcell death is due to the propagation of stress and shock waves in theskin generated by the rapid deceleration of the microparticles (McSloyet al. (2004)). The rapid rise time of these stress waves in the skin,and their magnitude both contribute to cell death and the results areconsistent with the findings reported by Doukas, A. G. & Flotte, T. J.(1996). Physical characterization and biological effects oflaser-induced stress waves. Ultrasound in Medicine and Biology,22(2):151-164. The effects of shock waves and particle penetration inthe skin on cell viability following gene gun delivery. Masters Thesis,Department of Engineering Science, University of Oxford.). Thismechanism of ballistic particle penetration killing cells negativelyaffects the ability of the direct and efficient delivery of genes anddrugs to the cells.

This cell death effect of ballistic particle delivery could be reducedby significantly decreasing the particle size to the nanometerregime-thereby reducing the stresses on the cells. However, anotherlimitation of the gene gun is that it is unsuitable in deliveringsub-micron sized particles to cells. This is illustrated by thefollowing. As reported (Kendall, M. A. F., Mitchell, T. J. &Wrighton-Smith, P. (2004) Intradermal ballistic delivery ofmicro-particles into excised human skin for drug and vaccineapplications. J Biomechanics, 37(11):1733-1741), and shown in FIG. 14,ballistic particle penetration is proportional to the particle impactparameter, pvr, which is the product of the particle density (φ,velocity (v) and radius (r). This parameter is also proportional to theparticle momentum per-unit-area, which has been shown to drive themechanism of particle penetration depth (Mitchell et al. (2003)). FromFIG. 14, we see a 1 μm radius gold particle (density 18000 kg/m′) wouldneed to impact the skin at ˜600 m/s in order to penetrate to reach cells˜20 μm into the skin.

Experimental results show that reducing the particle radius, say, by anorder of magnitude, to 100 nm, and placing it in a standard biolisticdevice leads to negligible particle impact in the skin. Indeed from FIG.14 we see delivery to a 20 μm depth would need an impact velocity of˜6000 m/s, which is impractical for two reasons: 1) these hypervelocityconditions can not be safely achieved with a system configured for humanuse (they are usually achieved with massive free-piston shock tunnels);2) even if 6000 m/s was obtained in the free-jet, a gas impingementregion above the skin would seriously decrease the particle velocity—itis possible that the particle would not even hit the skin at all.Interestingly if a method was conceived to safely and practicallydeliver nanoparticles to the skin at higher velocity (e.g., the statedcase of an 100 nm radius gold particle at a velocity of ˜6000 m/s), thecell death benefit of smaller scale would be offset by higher peakstresses-killing more cells—and higher strain rates that are likely tofurther “toughen” the skin, making delivery even more difficult.

In conclusion, these collective facts rule out the gene gun as a viableoption for delivering nanoparticles and therefore precludes it from manyof the developments in biomolecules, drugs and sensors at this scale.

The huge research effort in micro- and nanotechnologies providestremendous potential for simple and practical cell targeting strategiesto overcome many limitations of current biolistic (and other) celltargeting approaches. For example, FIG. 11(c) shows that the mostconceptually simple and appealing approach to gene delivery is thedirect injection of naked DNA to live cell nuclei at a sub-micrometerscale that does not adversely damage the cell (Luo, D. & Saltzman, W. M.(2000) Synthetic DNA delivery systems. Nature Biotechnology, 18:33-36).Cell death is minimized by both the sub-micrometer scale of the injectorand the low, quasi-static strain-rate of the probe (compared toballistic delivery) resulting in low stress distributions. Although thisis a very efficient gene and bioagent delivery route, the to drawback isthat such precise targeting by direct microinjection can only beachieved one cell at a time and with great difficulty to the operator invivo. Hence, the method is slow, laborious and impractical.

Researchers have overcome some of these disadvantages for transdermaldrug delivery by fabricating arrays of micrometer-scale projections(thousands on a patch) to breach the stratum corneum for the intradermaldelivery of antigens and adjuvants to humans and other mammals.

In the scientific literature, the first description of this techniqueappears to be the paper Microfabricated Microneedles: A Novel Approachto Transdermal Drug Delivery. S. Henry et al, J. Pharmaceutical Sci.vol. 87(8) p 922-925 (1998), with the accompanying patent of U.S. Pat.No. 6,503,231. The objective of U.S. Pat. No. 6,503,231 is to provide amicroneedle array device for relatively painless, controlled, safe,convenient transdermal delivery of a variety of drugs and forbiosampling. This is achieved by the microneedles breaching the tissuebarrier (e.g., for skin: the stratum corneum) and then the therapeuticor diagnostic material is injected through the hollow microneedles intothe tissue. Specifically, in claim 1 of U.S. Pat. No. 6,503,231, it isstated that the microneedles are to be hollow, with a length of 100 μm-1mm, and claim 3 states the width of 1 μm-100 μm, with subsequent claimsstating ways the hollow needles can be connected to reservoirs for theinjection of liquids, fabrication methods, materials and examples ofdrugs to be delivered. Thus, U.S. Pat. No. 6,503,231 describes a patchsuitable for delivering materials and/or energy across tissue barriers.The microneedles are hollow and/or porous to permit drug delivery atclinically relevant rates across skin or other tissue barriers, withoutdamage, pain, or irritation to the tissue.

Other related microneedle devices in the patent literature are U.S. Pat.Nos. 5,527,288 and 5,611,806. More recently published patentapplications on this topic are WO02/085446, WO02/085447, WO03/048031,WO03/053258 and WO02/100476A2.

These microneedles array patch technologies have achieved only limitedsuccess to date. Generally, there are a range of approaches configuredto breach the stratum corneum to allow an enhanced take-up of drug inthe viable epidermis. Although this has not been discussed in thepatents referred to above, based upon reported research on ballisticparticle delivery and cell death, the low strain rate of application,combined with the cases of smaller projections are likely to induce alower incidence of cell death near the tips, than ballisticmicroparticle to delivery. Also, unlike ballistic microparticledelivery, these projections are removed from the tissue-alleviating thepossibility of adverse effects of “carrier” materials delivered to thebody, long term.

However, unlike biolistic targeting (FIG. 11(b)), and the directinjection of cells (FIG. 11(c)), these microneedle arrays do not havethe advantage of readily and directly targeting inside the skin cells.This cellular/organelle targeting capability is key in a range ofexisting and potential methods of vaccination, gene therapy, cancertreatment and immunotherapy (Needle-free epidermal powder immunization.Chen et al, Expert Rev. Vaccines 1(3) p265-276 (2002)) and diagnostictechnologies.

Whilst U.S. Pat. No. 5,457,041 describes a patch for targeting cells,this is only suitable for use in vitro, and requires specializedapparatus to direct the micro-needles towards identified cells. Theapparatus uses a microscope, to allow an operator to locate the cells inthe sample tissue, and then direct the application of the micro-needlesappropriately. As a result, this makes the device unsuitable for use inclinical environments, and limits the ability of the device to elicit adesired biological response.

Therefore, there still remains a need to provide projection-basedtechnology which achieves a more accurately directed delivery of theactive agent or stimulus to the desired site of action surrounding orwithin cells, without appreciable damage to them.

BRIEF SUMMARY

In a first broad form the present invention provides a device fordelivery of material or stimulus to targets within a body to produce adesired response, the targets being at least one of cells of interest,cell organelles of interest and cell nuclei of interest, the deviceincluding a number of projections for penetrating a body surface, andwherein:

-   -   a) the number of projections is selected to produce a desired        response, the number being at least 500; and,    -   b) a spacing between projections is at least partially        determined based on an arrangement of the targets within the        body.

Typically the number of projections is selected by:

-   -   a) determining a likelihood of a projection targeting at least        one of the targets;    -   b) determining a number of targets to be targeted; and,    -   c) determining the number of projections using the determined        likelihood and the determined number of targets.

Typically the likelihood P_(contact) of a projection targeting a targetof interest is at least partially based on:

$\begin{matrix}{{1.\mspace{14mu} P_{contact}} = \frac{V_{tar}}{V_{layer}}} & \;\end{matrix}$

-   -   ii) where:        -   (a) V_(layer) is the volume of the layer containing targets,        -   (b) V_(tar) is the volume including the target of interest            to which material or stimulus can be delivered.

Typically the number of targets to be targeted depends on the number oftargets that need to be transfected to produce the desired response.

Typically the number of targets to be targeted is at least one of:

-   -   a) at least 10;    -   b) at least 100;    -   c) at least 1000;    -   d) at least 10000;    -   e) at least 100000;    -   f) at least 1000000; and,    -   g) at least 10000000.

Typically the number of projections is at least one of:

-   -   a) at least 10;    -   b) at least 100;    -   c) at least 1000;    -   d) at least 10000;    -   e) at least 100000;    -   f) at least 1000000; and,    -   g) at least 10000000.

Typically a maximum number of projections is based on at least one of:

-   -   a) the total surface area of the target site available;    -   b) a minimum projection spacing (S); and,    -   c) an upper limit in active material or stimulus to be delivered

Typically the projection spacing is based at least partially on at leastone of:

-   -   a) a size of the targets of interest; and,    -   b) a spacing between the targets of interest.

Typically the spacing between at least some of the projections isselected to avoid multiple projections targeting a single target ofinterest.

Typically the spacing between at least some of the projections isselected to be greater than a diameter of the targets of interest.

Typically the spacing between at least some of the projections isselected to be approximately equal to the spacing between the targets ofinterest.

Typically the spacing S between at least one of:

-   -   a) 1 μm≤S≤10000 μm; and,    -   b) 10 μm≤S≤200 μm.

Typically projection dimensions are based at least partially on anarrangement of targets within the body.

Typically at least some of the projections have a diameter of at leastone of:

-   -   a) less than the size of targets; and,    -   b) of the order of the size of targets within the targets.

Typically at least some of the projections have a projection length atleast partially based on a depth of the targets below a surface of thebody against which the device is to be applied in use.

Typically the projections include a support section and a targetingsection.

Typically the targeting section has a diameter of less than at least oneof:

-   -   a) 1 μm; and,    -   b) 0.5 μm.

Typically a length for the targeting section is at least:

-   -   a) less than 0.5 μm; and,    -   b) less than 1.0 μm; and,    -   c) less than 2.0 μm.

Typically a length for the support section is at least partially basedon a depth of the targets below a surface of the body against which thedevice is to be applied in use.

Typically the length for the support section is at least partiallydetermined in accordance with properties of a surface of the bodyagainst which the device is to be applied in use.

Typically at least one of a support section length and the number ofprojections is at least partially based on a likelihood of a projectionpenetrating the targets:

$P_{depth} = {\int_{{B - {Q\; \sigma}}\;}^{T + B - {Q\; \sigma}}{\frac{1}{\sigma \sqrt{2\pi}}e^{- {(\frac{x - D}{\sigma})}^{2}}}}$

where:

-   -   (a) σ is the standard deviation from a mean location, accounting        for the skin surface undulations.    -   (b) D is a distance of the targets below a surface of the body        against which the device is to be applied in use;    -   (c) Q is a number of standard deviations from a mean level of        the surface of the body at which the device comes to rest in        use;    -   (d) B is a length of the support section; and,    -   (e) T is a length of a targeting section.

Typically a length for the support section is at least one of:

-   -   a) for epidermal delivery <200 μm;    -   b) for dermal cell delivery <1000 μm;    -   c) for delivery to basal cells in the epithelium of the mucosa        600-800 μm; and,    -   d) for lung delivery of the order of 100 μm in this case.

Typically the length of the delivery end section is greater than thetarget dimension.

Typically at least some of the projections within a targetingconfiguration have different dimensions.

Typically the projections are solid.

Typically the projections are non-porous and non-hollow.

Typically at least part of at least some of the projections are coatedwith a bioactive material.

Typically at least part of at least some of the projections are coatedwith a non-liquid material.

Typically at least part of a targeting section of at least some of theprojections are coated.

Typically the coating is at least one of:

-   -   a) nanoparticles;    -   b) a nucleic acid or protein;    -   c) an antigen, allergen, or adjuvant;    -   d) parasites, bacteria, viruses, or virus-like particles;    -   e) quantum dots, SERS tags, raman tags or other nanobiosensors;    -   f) metals or metallic compounds; and,    -   g) molecules, elements or compounds.

Typically the device includes at least some uncoated projections tothereby stimulate or perturb the targets in use.

In one example, the device includes:

-   -   a) a flexible substrate; and,    -   b) a number of patches, each patch including a number of        projections for penetrating a body surface, the number of        patches being mounted to a flexible backing.

In a second broad form the present invention provides a method ofselecting constructional features for a device a for delivery ofmaterial or stimulus to targets within a body to produce to a desiredresponse, the targets being at least one of cells of interest, cellorganelles of interest and cell nuclei of interest, the device includinga number of projections for penetrating a body surface, and wherein themethod includes:

-   -   a) selecting the number of projections to produce a desired        response, the number being at least 500; and,    -   b) selecting a spacing between projections at least partially        based on an arrangement of the targets within the body.

In a third broad form the present invention provides a method offabricating a device for delivery of material or stimulus to targetswithin a body to produce a desired response, the targets being at leastone of cells of interest, cell organelles of interest and cell nuclei ofinterest, the device including a number of projections for penetrating abody surface, and wherein the method includes:

-   -   a) selecting the number of projections to produce a desired        response, the number being at least 500;    -   b) selecting a spacing between projections at least partially        based on an arrangement of the targets within the body; and,    -   c) fabricating the device using the selected number of        projections, and the selected spacing

In a fourth broad form the present invention provides a method oftreating a subject the method including using a device for delivery ofmaterial or stimulus to targets within the subject's body to produce adesired response, the targets being at least one of cells of interest,cell organelles of interest and cell nuclei of interest, the deviceincluding a number of projections for penetrating a body surface, andwherein:

-   -   a) the number of projections is selected to produce a desired        response, the number being at least 500; and,    -   b) a spacing between projections is at least partially        determined based on an arrangement of the targets within the        body.

In a fifth broad form the present invention provides apparatus fordelivery of material or stimulus to targets of interest within a body toproduce a desired response, the targets being at least one of cells ofinterest, cell organelles of interest and cell nuclei of interest, theapparatus including:

-   -   a) a structure;    -   b) a plurality of projections movably mounted to the structure        for penetrating a body surface;    -   c) an actuator for selectively releasing the plurality of        projections mounted on the movable structure from a retracted        position such that upon contact with the body surface the        plurality of projections enter the body.

Typically the plurality of projections are provided on a patch.

Typically the actuator includes:

-   -   a) a spring coupled to the structure and the at least one patch;        and,    -   b) a releasing means for releasing the spring, to thereby        release the plurality of projections from the retracted        position.

Typically the releasing means is a tensioned string for holding thespring in compression.

Typically the releasing means is manually operated.

Typically the apparatus includes a number of arms, each arm beingcoupled to a respective spring and including a first end pivotallymounted to the structure and a second end coupled to a respectiveplurality of projections, and wherein activation of the releasing meanscauses each of the arms to be released from a retracted position tothereby cause projections on the respective patch to enter the body.

Typically the arms are circumferentially spaced around a part of thestructure.

Typically the structure is flexible structure allowing the structure tobe guided to a desired location within the body.

Typically the releasing means includes an inflatable structure coatedwith the plurality of projections.

Typically:

-   -   a) the number of projections is selected to produce a desired        response, the number being at least 500; and,    -   b) a spacing between projections is at least partially        determined based on an arrangement of the targets within the        body.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

An example of the present invention will now be described with referenceto the accompanying drawings, in which:—

FIGS. 1A and 1B are schematic diagrams of an example of device fordelivery of material or stimulus to targets within a body;

FIG. 1C is a schematic diagram of an example of the device of FIG. 1A inuse;

FIGS. 1D and 1E are schematic diagrams of examples of projections usedin the device of FIG. 1A;

FIG. 2 is a flow chart of an example of the process of selecting deviceparameters;

FIGS. 3A and 3B are schematic diagrams of alternative examples of thedevice of FIG. 1A in use taking into account variations in surfaceproperties and target locations;

FIG. 4 is a flow chart of a second example of the process of selectingdevice parameters;

FIGS. 5A and 5B show examples of the relationship between the number ofprojections and total hits for targeting Langerhans cell nuclei andLangerhans cells respectively;

FIG. 6 shows an example of the relationship between the total number oftargeted LC and the patch surface area as a function of projectionspacing geometry;

FIG. 7 is an SEM photograph of an example of a constructed patch;

FIG. 8 shows an example of the Transfection Probability vs NeedleSpacing, for targeting of Langerhans cells;

FIGS. 9A to 9C show examples of projection viability against theprojection height, for variation in the skin surface level standarddeviation of 20 μm, 40 μm and 60 μm respectively;

FIG. 10 is a schematic diagram of an alternative example of a patch;

FIG. 11 illustrates a schematic cross-section of skin structure: (a)half-section scale of a typical smaller needle and syringe (diameter˜0.5 mm); (b) penetration of microparticles following biolisticdelivery; (c) idealized direct injection of a cell nucleus; (d) amicro-nanoprojection array;

FIGS. 12A and 12B illustrate the effects of relative humidity (A) andambient temperature (B) on to ballistic particle penetration into theskin;

FIG. 13A is a graph showing the relationship between percentage celldeath (membrane rupture) and particle density (McSloy (2004) MA Thesis,University of Oxford);

FIG. 13B is a diagram showing how the data for FIG. 13A was retrieved(McSloy (2004) MA Thesis, University of Oxford);

FIG. 13C is a graph showing membrane rupture versus distance of cellpathway;

FIG. 14 illustrates the particle penetration parameter (pvr) vs.penetration depth obtained by the ballistic delivery of goldmicroparticles into skin (Kendall et al. (2004), Journal ofBiomechanics);

FIG. 15 shows examples of organelles within the cell(http://niko.unl.edu/bs101/notes/chapter4.html);

FIG. 16 is a schematic diagram of the skin structure;

FIG. 17 is an example of a histology micrograph of human skin with aLangerhans Cell (L) and Keratinocyte (K) stained. From Roitt et al, theheight of which is approximately 50 μm;

FIG. 18 illustrates the distribution of Langerhans Cells in a mouse ear(Kendall M. A. F., Mulholland W. J., Tirlapur U. K., Arbuthnott E. S.,and Armitage, M. (2003) “Targeted delivery of micro-particles toepithelial cells for immunotherapy and vaccines: an experimental andprobabilistic study”, The 6th International Conference on CellularEngineering, Sydney, August 20-22);

FIGS. 19A and 19B illustrate (A) a sample of canine buccal mucosaltissue and (B) the structure of the mucosa, Mitchell, T (2003) DPhilThesis, Department of Engineering Science, University of Oxford;

FIG. 20 illustrates the shape and dimensions of examples nanoneedles;

FIG. 21 illustrates the maximum needle length vs. nanoneedle diametercalculated using expression (3) and the Young's Modulus (E) values forGold, Titanium, ZnO, PGCA, Silver and Tungsten (respectively: 77.2, 116,111.2 and 7 GPa);

FIGS. 22A-22C are a Transmission Electron Micrographs (TEM) of amicro-nanoprojection electropolished from tungsten at (A) ×500magnification, (B) a bright field ×33000 magnification and (C) a darkfield ×33000 magnification;

FIGS. 23A and 23B illustrate fluorescent microscope images of tungstenrods, (A) uncoated and (B) coated in eGFP plasmid DNA immersed in anethidium bromide solution;

FIGS. 24A-24D are examples of an optically sectioned Multi-PhotonMicroscopy (MPM) images of the agar after insertion of a DNA coatedtungsten probe (A) on the surface (B) at a depth of 13 μm (C) at a depthof (32 μm). Also shown in (D) is an optical section at 32 μm of agar gelfollowing insertion of a probe without a DNA coating;

FIG. 25 illustrates a photomicrograph of a micro-nanoprojectionelectropolished from tungsten used in skin tissue indentation experimentwith scale bar;

FIG. 26 illustrates two sample load-displacement curves in freshlyexcised mouse ear tissue obtained with the micro-nanoprojection shown inFIG. 25;

FIG. 27 is a plan view diagram of possible alternative geometry of thenanoneedle;

FIG. 28 illustrates an example of a nanoneedle array or patch device;

FIG. 29 is a schematic diagram of an example of the nanoneedle arrayproduced with 2PLSM;

FIGS. 30A-30C illustrate sequences for producing a mask;

FIG. 31 is a schematic diagram of an example of a “Stepped” nanoneedle;

FIGS. 32A and 32B illustrate examples of an intradermal application ofnanoneedle patches;

FIG. 33 is a schematic diagram of an example of an applicator, fittedwith the patch for mucosal delivery;

FIG. 34 illustrates the major respiratory organs; and,

FIGS. 35A-35C are schematic diagrams of an example of a deployable patchstructure for targeting the lower airway and lung.

DETAILED DESCRIPTION

An example of a device for delivering material or stimulus targetswithin a body will now be described with reference to FIGS. 1A to 1E.

In this example, the device is in the form of patch 100 having a base120 and a number of projections 110. The base 120 and projections 110may be formed from any suitable material, as will be described in moredetail below, but in one example, are formed from a silicon typematerial, allowing the device to be fabricated using fabricationprocesses such as vapor deposition, silicon etching, Deep Reactive IonEtching (DRIE), or the like.

In the example shown, the device has a width W and a breadth B with theprojections 110 being separated by spacing S.

In use, the patch 100 is positioned against a surface of a subject,allowing the projections to enter the surface and provide stimulus ormaterial to targets therein. An example of this is shown in FIG. 1C.

In this example, the patch 100 is urged against a subject's skin showngenerally at 150, so that the projections 110 pierce the Stratum Corneum160, and enter the Viable Epidermis 170 to reach targets of interest,shown generally at 180.

Examples of suitable projections are shown in more detail in FIGS. 1Dand 1E.

In each example, the projection generally includes a targeting section111, intended to deliver the material or stimulus to targets within thebody, and a support section 112 for supporting the targeting section111.

In the example of FIG. 1D, the projection is formed from a conicallyshaped member, which tapers gradually along its entire length. In thisexample, the targeting section 111 is therefore defined to be the partof the projection having a diameter of less than d₂.

As an alternative example however, the structure of the projection mayvary along its length to provide a targeting section 111 with a designedstructure. In this example, the targeting section 111 is in the form ofa substantially cylindrical shape, such that the diameter d₁ isapproximately equal to the diameter d₂.

In either case, the support section has a length a, whilst the targetingsection 111 has a length l. The diameter of the tip is indicated by d₁,whilst the diameter of the support section base is given by d₃.

In use, the device is intended to deliver material or stimulus tospecific targets within the body. Thus, rather than just operating todeliver material to, for example, the blood supply, or tissue within thebody, the device is configured so as to ensure material or stimulusreaches specifically selected targets such as cells, cell organelles,cell nuclei, or the like. Furthermore, the device is designed to achievethis without requiring specific directional control of deviceapplication so as to ensure the projections reach the targets. In otherwords, the device is intended to ensure successful delivery of materialor stimulus to specific targets within a subject, without requiring thatthe projections are aimed at the specific targets, but rather byallowing general placement in a suitable region. Thus, for exampleplacement may to be as simple as placement anywhere on the user's skinin order to target Langerhans cells of the device on the subject.

To achieve this, the device is provided with a particular configurationto ensure successful targeting. Accordingly, it is generally necessaryto select patch parameters, such as the number of projections N, andspacing S between projections, to be dependent upon the intended use ofthe device. A mechanism for achieving this will now be described withreference to FIG. 2.

In this example, at step 200 it is necessary to determine an arrangementof desired targets. This may be achieved in any one of a number of waysand will depend on the nature of the targets. Thus, for example if thetargets are a specific type of cell, cell nuclei, or cell organelle,this information can be determined from literature or studies detailingthe typical location of cells, or other targets, within the body.

At step 210 a number of projections required to elicit a desiredresponse is determined. This can depend on a range of factors, such asthe ability of projections to reach the desired targets, the ability ofthe projections to deliver material or stimulus to the targets, as wellas the ability of the targets to elicit a response. Thus, for example,non-uniform distribution of targets within the body means that it is notpossible to assume that each projection will deliver material orstimulus to a desired target during use of the device.

The number of projections may be determined in any one of a number ofways. Thus, for example, this can include selecting a number ofprojections from a predetermined list outlining the number ofprojections required for specific uses. However, if the number has notpreviously been determined, for example, if the target has notpreviously been used, then some form of analysis is typically required.

In one example, this is achieved by analyzing the distribution oftargets within the body and then determining a likelihood of any oneprojection reaching a target. An indication of the number of targets towhich stimulus or material must be delivered can then be used todetermine an indication of the number of projections required.

As will be described in more detail below, in general a desired responsecannot be obtained with less than 500 projections. More typically atleast 750 projections are required. However, in some instances, evenmore projections such as at least 1000, 2000, 5000, 7500, 10,000,100,000, 1,000,000 or even 10,000,000 may be used, and specific exampleswill be described in more detail below.

Once a number of projections has been selected, a projection spacing isdetermined at least partially based on the target arrangement at step230.

The spacing may be determined in any one of a number of ways, buttypically includes setting a lower spacing limit to ensure that only asingle projection delivers material or stimulus to a single target. Themaximum spacing S is typically set based on the required patch size(B×W) and/or the spacing between targets. It will be appreciated thatwhilst the example shown is rectangular, alternative shapes, such ascircular, elliptical, hexagonal, or the like, may be used and that theuse of a rectangular patch is for the purpose of example only.

At step 240 a patch is fabricated in accordance with the selected patchparameters, including the number of projections N and the spacing S.Fabrication may be achieved in a number of ways, as will be described inmore detail below.

By selecting at least a number of projections N required to elicit adesired response, this allows a patch to be provided with sufficientprojections to ensure that a desired response is achieved by delivery ofmaterial or stimulus to specific targets. Furthermore, by utilizing aprobabilistic analysis, this technique ensures that the requiredtargeting will be achieved without requiring the individual projectionsto be aimed at the individual targets. Thus, in contrast to other priorart techniques, the patch 100 may simply be inserted into a body at ageneral location, and does not need specialized apparatus to direct theprojections towards specific cells or other targets within the body.

A more detailed example of the process will now be described. For thepurpose of this example the patch configuration, and in particular theinsertion of the patch into the body is as shown in FIG. 3A and FIG. 3B.In particular, this example is modified to take into account variationsand undulations in the surface of the body, as well as variations intarget depth.

In this example, the patch 100 is urged against the surface 300 of theStratum Corneum 310. The surface 300 includes undulations, resulting ina mean surface level 320 shown by dotted to lines, with the patch base120 resting against the surface 300 at a distance y above the mean level320.

The projections 110 enter the Viable Epidermis 330 to deliver materialor stimulus to targets 340, which are generally arranged in a layer 350,referred to as the target layer. The Dermis is also shown at 360 in thisexample.

In the example of FIG. 3A the targets 340 are provided in a single layerwith each target being approximately a constant depth D_(c) below theStratum Corneum 310. In this example, the layer height h_(layer) istherefore approximately equal to the diameter of the targets d_(c), withthe targets separated by a spacing S_(c). It would be appreciated bypersons skilled in the art that in this instance the targets maytherefore be Langerhans Cells, or the like.

In the example of FIG. 3B, the targets 340 are dispersed verticallythrough the Viable Epidermis 330, so that the target layer 350 has agreater height h_(layer) than in the previous example. Additionally, inthe example, the depth of the targets is calculated on the basis of themean layer depth, as shown.

An example of the process for selecting a device configuration to takeinto account the arrangements in FIGS. 3A and 3B will now be describedin more detail with reference to FIG. 4.

At step 400 a desired target and corresponding target arrangement isdetermined.

The target selected will depend on the intended application. Thus, forexample, the target may be cells, cell nuclei or cell organelles.Additionally, different types of cells may need to be targeted. Thus,for example, cells such as Langerhans Cells may be stimulated forproviding an immunological response, whereas cells such as squamous orbasal cells may need to be targeted to treat cell carcinoma. An exampleof other potential targets will be described in more detail below.

Determining the target arrangement typically involves determiningparameters relating to the target such as target depth D_(c), targetdiameter d_(c), layer height and target spacing S_(c). Thus thesecorrespond to the parameters outlined above with respect to FIGS. 3A and3B.

At step 410 the diameter of the targeting section 111 for at least someof the projections is determined.

The diameter of the targeting section is typically based on the size ofthe target. Thus, for example, the diameter of the targeting sectiondoes not usually exceed the scale of the target, as this may lead totarget necrosis. In general this leads to an upper limit for targetingsection diameters of:

d ₁≤1 μm and d ₂≤2 μm.

However, it will be appreciated that smaller diameters such as 500 nm,or below, may be used, as described in specific examples below.Additionally or alternatively, it may be desirable to includeprojections having a larger diameter, for example to cause cellnecrosis. In one example, at least some of the projections have adiameter greater than 1 μm, which can be used to induce bystanderresponses. In other examples, all of the projections have a diametergreater than 1 μm to thereby kill targets.

At step 420 the projection length is determined. In one example, theprojection length is based on the depth of the target layer D_(c) andthe layer height h_(layer). Thus, the length of the supporting sectionof a can be selected so that the targeting section 111 at least reachesthe target layer within the body, but typically does not extend a largedistance beyond the target layer. In this example, the length a of thesupport section 112 is typically given by:

(D _(c) +h _(layer)/2)≥a≥(D _(c) −h _(layer)/2)

Similarly the length l of the targeting section 111 is typicallyselected to be at least equal to the layer height h_(layer) to ensurepenetration of targets within the layer 340, so that:

l≥h _(layer)

As shown in the examples of FIGS. 3A and 3B however the skin of the bodyis not generally flat but is undulating. As a result, this means thatthe base 120 of the device 100 generally sits a distance y above themean skin level 320. To take this into account, the length of thesupport section 112 may be increased such that.

(y+D _(c) +h _(layer)/2)≥a≥(y+D _(c) −h _(layer)/2)

Alternatively, a probabilistic analysis can be used to determine thelikelihood of a viable to projection of a given length reaching thetargets with the body. This will depend on a number of factors, examplesof which, for the targeting of Langerhans cells (LC), include:

-   -   The surface of the skin is normally distributed    -   It is assumed that the LC reside exactly 17 μm below the surface        just above the basal layer (Arbuthnott, 2003, Emislom et al,        1995), in the case of the Balb/c mouse ear. In reality, there        may be a small variation in depth of LC. For example, the depth        of LC varies significantly from site to site within a given        animal of human model, and there is typically variation in the        depth of LC between models—for example the depth of LC in humans        is greater than in mice.    -   The patch typically comes to rest two standard deviations away        from the mean skin level    -   The needles have a viable tip length of 20 μm (i.e., l, (defined        as 111) is 20 μm. Penetration by any other part of the needle        other than this tip causes cell death.    -   The skin's surface is normally distributed with mean 0 and        standard deviation σ.

Within the model:

-   -   The Langerhans cells lie D_(c) microns directly below the skin's        surface.    -   The patch comes to rest Q standard deviations from the skin's        mean level.    -   The needles have body (unviable) length support section a and        tip (viable) length l.

In this example, the likelihood of viable targeting of targets at thedefined depths (P_(depth)) is given by:

$\begin{matrix}{P_{depth} = {\int_{{a - {Q\; \sigma}}\;}^{l + a - {Q\; \sigma}}{\frac{dx}{\sigma \sqrt{2\pi}}e^{- {(\frac{x - D_{c}}{\sigma})}^{2}}}}} & (1)\end{matrix}$

where:

-   -   σ is the standard deviation from a mean location, accounting for        the skin surface undulations.    -   D_(c) is a distance of the cells of interest below a surface of        the body against which the device is to be applied in use;    -   Q is a number of standard deviations from a mean level of the        surface of the body at which the device comes to rest in use;    -   a is a length of the support section; and,    -   l is a length of a targeting section.

In this example it is assumed that the skin surface level is normallydistributed on a local scale, which is typically a safe assumption basedon histology samples, although if the area inspected is too large,global curvature invalidates the assumption. At first instance, it isassumed that the patch comes to rest at a distance y of two standarddeviations away from mean skin level.

The use of such a model allows support section and targeting sectionlengths a, l to be selected to ensure a predetermined probability of thetargeting section 111 successfully reaching the targets. In one example,the probability is selected to be one, to ensure successful delivery ofthe material or stimulus. Alternatively, lower probabilities may beselected, with this being taken into account in determining the numberof projections provided, as will be described in more detail below.

At step 430, the likelihood of a single targeting event is determined.In particular, this is the likelihood of a projection, assuming theprojection reaches the target layer 350, of delivering material to orstimulus to a respective target 340.

In one example, it is assumed that the targeting section 111 iseffectively a point diameter cylinder that extends into the target layer350. In this instance the likelihood of a single targeting event isgiven by the volume of the target divided by the volume of the layer.

$\begin{matrix}{P_{contact} = \frac{V_{tar}}{V_{layer}}} & (2)\end{matrix}$

where:

-   -   V_(layer) is the volume of the layer containing cells of        interest,    -   V_(tar) is the volume including the target to which material or        stimulus can be delivered.

In this regard it will be appreciated that the volume to which stimulusor material may be to delivered may be significantly greater than thesize of the physical target itself, depending on the delivery mechanismused. Thus, for example, if material to be delivered to a cell isabsorbed when placed within the vicinity of the cell, then the targetvolume V_(tar) will be larger than the volume of the cell.

However, in a more detailed example, equation (2) still holds, but itcan be assumed that the projection has its own volume (V_(pro)), and inthis case the probability of contact is modified to change V_(tar) totake into account the additional probe volume. In one example, if theprojection is as shown in FIG. 1E, in which d₁=d₂, the volume of thetargeting section within the target layer is given by:

$\begin{matrix}{V_{tar} = {\sum\limits_{i = 1}^{N_{tar}}( {( {{2d_{1}} + d_{c}} )^{2}d_{c}} )}} & (3)\end{matrix}$

where:

-   -   N_(tar) is the number of targets, with diameter d_(c).

Equation (3) can be modified to take into account variations in targetsize and shape, and probe size and shape. When d₁ and d_(c) areconstant, equation (3) becomes:

V _(tar) =N _(Tar)(2d ₁ +d _(c))² d _(c)  (4)

So for a region of target layer of size z×z, where a projection reachesthrough the target layer, and containing a number of targets N_(tar),the probability is given by:

$\begin{matrix}{P_{{contact}\;} = \frac{{N_{tar}( {{2d_{1}} + d_{c}} )}^{2}d_{c}}{z^{2}h_{layer}}} & (5)\end{matrix}$

The above example assumes that the projection reaches fully through thetarget layer 350 (i.e., P_(depth)−1). However, this may not occur, andaccordingly the above described equation can be utilized to take intoaccount the probability of the projection reaching the relevant celllayer, as described in equation (1) above.

So the general expression for the probability of a projection targetingthe targets of interest (e.g., key cells, nuclei, etc.), defined asP_(tar) is:

P _(tar) =P _(contact) ×P _(depth)  (6)

It will be appreciated that in the event that the projection lengths a,l are selected such that P_(depth)=1, then P_(tar)=P_(contact).

At step 440 the number of targeting events required is determined. Thisis typically determined on the basis of studies performed indicating thenumber of targets to which material or stimuli must be delivered inorder for a biological response to be affected. Often, this isdetermined by parametric empirical studies.

For example, when it is desired to deliver DNA to transfect Langerhanscells, at least one cell must be targeted. However, it is believed thatto ensure a successful biological response, at least 10 cells, and morepreferably at least 100 cells and up to or over 1,000 cells aretargeted. It will be appreciated however that for different deliverymechanisms a different number of targets may be desirable.

At step 450, having determined the number of targeting events required(N), it is possible to use this and the probability of a singletargeting event to determine a number of projections required, which isgiven by:

N=T/P _(tar)

where:

T is the total number of delivery events required to produce the desiredresponse.

Often in immunotherapeutic and drug applications (including vaccines), Tis defined as a range, that can vary significantly between applicationand/or model (i.e., animal or human).

Specific examples of this are shown in FIGS. 5A and 5B, for thetargeting of Langerhans cell nuclei and Langerhans cells respectively.

In these examples it is assumed that:

-   -   A Langerhans Cell (LC) penetrated by one needle remains viable.        If it is penetrated by any more than one projection cell death        occurs.    -   When a projection contacts the target site (e.g., cell nucleus        or cytosol), the desired biological “event” occurs each time. In        reality, there is probability of the event happening. For        example, if cell transfection is required, then the probability        of this event through the delivery of DNA to nucleus via a        projection is ˜0.9, whereas the probability of the same event        from the coated projection entering only the cytoplasm is ˜0.1        (Nagasaki 2005).    -   A dead cell cannot be transfected.    -   Penetration by a needle with a diameter greater than 1 μm will        cause cell death.    -   LC are assumed as spherical with 10 μm diameter.    -   Nuclei of LC are assumed to be spherical, with a diameter of 4        μm (Arbuthnott, 2003).    -   All LC lie just above the basal layer between the epidermis and        dermis (as reported by Kendall et al. (2003).

LC are oval with dimensions circa 11.25×6 μm but to simplify the model,the average of these two figures is taken as the diameter of 10 μm. Inone example of young Balb/C mice the LC density is 895 cells/mm² (Choi.et al 1987). With LC uniformly distributed in the suprabasal region(Numahara et al. 2001) this gives a center to center spacing ofapproximately 30 microns, justifying the spacing assumption. Forsimplicity, it is assumed that the LC spacing of 1000 cells/mm², givinga planar spacing between cell centers of 32 μm.

FIG. 5A shows the relationship between the number of projections andtotal hits is given for the patches 100 described above, with a range ofdepth probabilities (P_(depth)), for targeting Langerhans cell nuclei.

FIG. 5B shows the relationship between the number of projections andtotal hits is given for the patches 100 described above, with a range ofdepth probabilities (i.e., the probability of a viable targeting of thetarget layer 350), for targeting Langerhans cells.

Typically this requires at least 500 projections and more typically atleast 1,000 projections for targeting LC and 10,000 projections fortargeting LC nuclei. Specific examples of this will be described in moredetail below.

At step 460 the projection spacing (S) is then determined based on thetarget diameter and target spacing. It is usual to assume that no morethan one projection should enter a single cell this; will typically leadto cell death. Accordingly, it is typical to select a projection spacingwhich is at least greater than a cell diameter, such that:

S≥d _(c)

More preferably it is typical to select a size for the projectionspacing based on a preferred overall patch size. In particular, it ispreferred that patches are made below a certain upper limit defined bypractical utility to the targeting site of the patient or animal.

For example, if the targeting site is the skin of the human abdomen,then the surface area could be approaching the surface area of theabdomen (e.g., ˜400 cm², or 20×20 cm, say). It would not be practical toachieve such larger surface areas over surfaces with “bulk” curvature(such as the abdomen example, or arm) with one large rigid patch. In oneexample, surface area can be achieved using the patch shown in FIG. 10.In this example, the patch 100 is formed from several smaller patches1000 provided on a flexible backing material 1010. The patch assemblycould be extended to wrap around the patient site 1020.

In any event, an upper limit on the spacing S is typically selected toensure that the desired number of projections fit on a patch of thedesired size.

A specific example of this is shown in FIG. 6, in which the relationshipbetween the total number of targeted LC (T) and the patch surface area(mm²) as a function of spacing geometry, is shown. In this example, theprobability of penetration to the cell depth is assumed to be one (i.e.,P_(depth)=1) for simplicity.

Irrespective of this, the projection spacing S is typically of the orderof spacing of the targets S_(c).

Once the spacing is selected the patch can be fabricated at step 470. Ingeneral, this will typically require considerations are taken intoaccount to ensure the projections are sufficiently robust to withstandpenetration of the Stratum Corneum and Viable Epidermis withoutbreaking. Example manufacturing processes will be described in moredetail below, and an example of a constructed patch 100 is shown in FIG.7. In this example, the device has 9795 projections, with a spacing of70 μm (S), over a surface area of 48 mm².

In general, a number of factors regarding the patch fabrication shouldbe noted.

Typically the projections are solid, non-porous and non-hollow. The useof solid projections enhances the projection strength, thereby reducingthe likelihood of projection breakage, which in turn helps maximizesuccessful delivery of material or stimulus to the targets. Also, solidprojections simplify device fabrication processes, allowing for theproduction of cheaper patches than for the case of porous or hollowprojections. This in turn further enhances the suitability of the patchfor use in medical environments.

To achieve delivery of material, it is typical to coat at least thetargeting section 111 with a non-liquid bioactive material, such as DNA.

The patch 100 may also be fabricated to perturb targets so as to induce“bystander” interactions. This may be used, for example, so that celldeath is used to release molecules to activate nearby targeted cells.This can be achieved in a number of manners, such as by providing amixture of coated and uncoated projections, as well as by providingprojections of differing dimensions including clusters of more than 1projection to target individual cells and/or larger scale tips to damagecells or other targets, upon insertion/residence/retrieval.

Specific Examples

A number of specific examples will now be described.

Transfection Probability

In this example, which focuses on the transfection of Langerhans cells,a number of additional practical considerations may also be taken intoaccount.

In particular, if the model of a point targeting section with no radiusis used, this predicts an idealized projection spacing S=6.5 μm, as usedin the example of FIG. 8. In this case, the LC diameter is ˜10 μm, sothat to satisfy the requirements set out above, namely that S≥d_(c), soS≥10 μm.

Also, for structural reasons, the diameter of the base of theprojections d₃ is likely to be above 6.5 μm, and a “clearance” will beneeded between each projection, so for practical reasons, the minimumprojection spacing (S) is at least 10 μm.

FIG. 8 shows an example of the Transfection Probability vs NeedleSpacing, for targeting of Langerhans cells with a spacing of 32 μm. TheS≥d_(c) criterion and practical considerations of minimum size of thebase d₃ suggest the spacing is to at least be 10 μm, as discussed above.

Needle Length Optimization

For this example, the sensitivity of needle length to standard deviationof the skin surface is shown in FIG. 9A to 9C. In particular, these showthe fraction of projections (P_(depth)) that are smaller than 1 μm indiameter and penetrate to 17 μm in the epidermis (the depth of LC),against the projection height, for skin level standard deviation of 20μm, 40 μm and 60 μm respectively.

It is clear from this, that the magnitude of skin undulations has astrong influence over the choice of optimum projection length.

Minimum Number of Projections

In one example, for targeting a single LC nucleus the likelihood ofcontact of a projection is given by P_(contact)=0.031, using equation(2) above, and data regarding LC nuclei size, set out in more detailbelow. The value of P_(depth) defined in equation (1), above has a rangeof 0-1, and with N=500, P_(depth)=0.064.

Utilizing this, it can be seen that a viable range of lengths result intargeting of a single cell, as shown below.

SD (roughness) Needle length needed FIG. (μm) (T = 1; N = 500) 9A 20 >80 μm. 9B 40 >130 μm. 9C 60 >160 μm 

All of these cases in the table above are well away from the optimallengths for the projections (i.e., where P_(depth) is a maximum in FIGS.9A-9C), so they do represent a “poor” case scenario, where the device isnot tuned well for the target (device tuning would be improved byreducing the needle length to closer to the optimal regions shown inFIG. 9A-9C, for example). However, the target of LC nuclei is very welldefined. So, on balance, a minimum of 500 projections is needed beforethere is a reasonable statistical chance of just one targeting event.

It will be appreciated that it is typical however to deliver to morethan one target, and accordingly, it is typical for a greater number ofprojections to be provided.

For example, the direct targeting of 2 LC nuclei (i.e., T=2) would beachieved with the device configurations in the table above with 1000projections (i.e., N=1000).

Similarly, the direct targeting of 10 LC nuclei (i.e., T=10) would beachieved with the device configurations in the table above with 5000projections (i.e., N=5000).

In the case of targeting situations with a lower P_(contact), forinstance in targeting a sparsely-populated dermal dendritic cellphenotype (not in a tightly defined layer like LC), greater than theminimum of 500 projections would be needed for at least one singletargeting event. For example, if P_(contact) is 1/10 of the stated LCcase (i.e., P_(contact)=0.0031), then applying a similar analysis, andadjusting for the deeper location of cells to maintain P_(depth)=0.064,then 5000 projections would be needed for the one targeting event.

Again, it will be appreciated that it is typical however to deliver tomore than one target, and accordingly, it is typical for a greaternumber of projections to be provided.

Maximum Number of Projections

The upper limit of projections is typically defined by a range ofparameters. These include the total surface area of the target siteavailable, and the minimum projection spacing (S). For example, inpreviously stated case of a human abdomen, with the patch assemblywrapped around to the back (i.e., ˜800 cm²), and the minimum spacing fortargeting cells (S=10 μm) results in 8,000,000 projections.

Another consideration is the payload to be delivered to the target site,where for a given application there is an upper limit in active materialor stimulus to be delivered. Here, if a given mass of active material iscoated to a single projection, then the total number of projectionswould be selected such that the total payload is less than this upperlimit.

In any event, it will be appreciated from the above that it is typicalto use at least 500 projections, but more typically at least 750, 1000,2000, 5000, 7500, 10,000, 100,000 projections, and even as many as10,000,000 projections.

Delivery

Illustrative stimuli or material that can be delivered with the deviceof the present invention include any or more of: small chemical orbiochemical compounds including drugs, metabolites, amino acids, sugars,lipids, saponins, and hormones; macromolecules such as complexcarbohydrates, phospholipids, peptides, polypeptides, peptidomimetics,and nucleic acids; or other organic (carbon containing) or inorganicmolecules; and particulate matter including whole cells, bacteria,viruses, virus-like particles, cell membranes, dendrimers and liposomes.

In some embodiments, the stimulus or material is selected from nucleicacids, illustrative examples of which include DNA, RNA, senseoligonucleotides, antisense oligonucleotides, ribozymes, smallinterfering oligonucleotides (siRNAs), micro RNAs (miRNAs), repeatassociated RNAs (rasiRNA), effector RNAs (eRNAs), and any otheroligonucleotides known in the art, which inhibit transcription and/ortranslation of a mutated or other detrimental protein. In illustrativeexamples of this type, the nucleic acid is in the form of an expressionvector from which a polynucleotide of interest is expressible. Thepolynucleotide of interest may encode a polypeptide or an effectornucleic acid molecule such as sense or antisense oligonucleotides,siRNAs, miRNAs and eRNAs.

In other embodiments, the stimulus or material is selected from peptidesor polypeptides, illustrative examples of which include insulin,proinsulin, follicle stimulating hormone, insulin like growth factor-1,insulin like growth factor-2, platelet derived growth factor, epidermalgrowth factor, fibroblast growth factors, nerve growth factor, colonystimulating factors, transforming growth factors, tumor necrosis factor,calcitonin, parathyroid hormone, growth hormone, bone morphogenicprotein, erythropoietin, hemopoietic growth factors, luteinizinghormone, glucagon, glucagonlike peptide-1, anti-angiogenic proteins,clotting factors, anti-clotting factors, atrial natriuretic factor,plasminogen activators, bombesin, thrombin, enkephalinase, vascularendothelial growth factor, interleukins, viral antigens, non-viralantigens, transport proteins, and antibodies.

In still other embodiments, the stimulus or material is selected fromreceptor ligands. Illustrative examples of receptors include Fcreceptor, heparin sulfate receptor, vitronectin receptor, Vcam-1receptor, hemaglutinin receptor, Pvr receptor, Icam-1 receptor,decay-accelerating protein (CD55) receptor, Car(coxsackievirus-adenovirus) receptor, integrin receptor, sialic acidreceptor, HAVCr-1 receptor, low-density lipoprotein receptor, BGP(biliary glycoprotien) receptor, aminopeptidease N receptor, MHC class-1receptor, laminin receptor, nicotinic acetylcholine receptor, CD56receptor, nerve growth factor receptor, CD46 receptor,asialoglycoprotein receptor Gp-2, alpha-dystroglycan receptor,galactosylceramide receptor, Cxcr4 receptor, Glvr1 receptor, Ram-1receptor, Cat receptor, Tva receptor, BLVRcp1 receptor, MHC class-2receptor, toll-like receptors (such as TLR-1 to -6) and complementreceptors.

In specific embodiments, the stimuli or material are selected fromantigens including endogenous antigens produced by a host that is thesubject of the stimulus or material delivery or exogenous antigens thatare foreign to that host. The antigens may be in the form of solublepeptides or polypeptides or polynucleotides from which an expressionproduct (e.g., protein or RNA) is producible. Suitable endogenousantigens include, but are not restricted to, cancer or tumor antigens.Non-limiting examples of cancer or tumor antigens include antigens froma cancer or tumor selected from ABL1 proto-oncogene, AIDS relatedcancers, acoustic neuroma, acute lymphocytic leukemia, acute myeloidleukemia, adenocystic carcinoma, adrenocortical cancer, agnogenicmyeloid metaplasia, alopecia, alveolar soft-part sarcoma, anal cancer,angiosarcoma, aplastic anemia, astrocytoma, ataxia-telangiectasia, basalcell carcinoma (skin), bladder cancer, bone cancers, bowel cancer, brainstem glioma, brain and CNS tumors, breast cancer, CNS tumors, carcinoidtumors, cervical cancer, childhood brain tumors, childhood cancer,childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma,choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia,colorectal cancers, cutaneous T-cell lymphoma, dermatofibrosarcomaprotuberans, desmoplastic small round cell tumor, ductal carcinoma,endocrine cancers, endometrial cancer, ependymoma, oesophageal cancer,Ewing's Sarcoma, Extra-Hepatic Bile Duct Cancer, Eye Cancer, Eye:Melanoma, Retinoblastoma, Fallopian Tube cancer, Fanconi anemia,fibrosarcoma, gall bladder cancer, gastric cancer, gastrointestinalcancers, gastrointestinal-carcinoid-tumor, genitourinary cancers, germcell tumors, gestational-trophoblastic-disease, glioma, gynecologicalcancers, haematological malignancies, hairy cell leukemia, head and neckcancer, hepatocellular cancer, hereditary breast cancer, histiocytosis,Hodgkin's disease, human papillomavirus, hydatidiform mole,hypercalcemia, hypopharynx cancer, intraocular melanoma, islet cellcancer, Kaposi's sarcoma, kidney cancer, Langerhan's cell histiocytosis,laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lipcancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma,Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breast cancer,malignant-rhabdoid tumor of kidney, medulloblastoma, melanoma, Merkelcell cancer, mesothelioma, metastatic cancer, mouth cancer, multipleendocrine neoplasia, mycosis fungoides, myelodysplastic syndromes,myeloma, myeloproliferative disorders, nasal cancer, nasopharyngealcancer, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegenbreakage syndrome, non-melanoma skin cancer, non-small-cell-lung-cancer(NSCLC), ocular cancers, esophageal cancer, oral cavity cancer,oropharynx cancer, osteosarcoma, ostomy ovarian cancer, pancreas cancer,paranasal cancer, parathyroid cancer, parotid gland cancer, penilecancer, peripheral-neuroectodermal tumors, pituitary cancer,polycythemia vera, prostate cancer, rare cancers and associateddisorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma,Rothmund-Thomson syndrome, salivary gland cancer, sarcoma, schwannoma,Sezary syndrome, skin cancer, small cell lung cancer (SCLC), smallintestine cancer, soft tissue sarcoma, spinal cord tumors,squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma,testicular cancer, thymus cancer, thyroid cancer,transitional-cell-cancer-(bladder),transitional-cell-cancer-(renal-pelvis-/-ureter), trophoblastic cancer,urethral cancer, urinary system cancer, uroplakins, uterine sarcoma,uterus cancer, vaginal cancer, vulva cancer, Waldenstrom'smacroglobulinemia, Wilms' tumor. In certain embodiments, the cancer ortumor relates to melanoma. Illustrative examples of melanoma-relatedantigens include melanocyte differentiation antigen (e.g., gp100, MART,Melan-A/MART-1, TRP-1, Tyros, TRP2, MC1R, MUC1F, MUC1R or a combinationthereof) and melanoma-specific antigens (e.g., BAGE, GAGE-1, gp100In4,MAGE-1 (e.g., GenBank Accession No. X54156 and AA494311), MAGE-3, MAGE4,PRAME, TRP2IN2, NYNSO1a, NYNSO1b, LAGE1, p97 melanoma antigen (e.g.,GenBank Accession No. M12154) p5 protein, gp75, oncofetal antigen, GM2and GD2 gangliosides, cdc27, p21ras, gp100^(Pmel117) or a combinationthereof. Other tumor-specific antigens include, but are not limited to:etv6, aml1, cyclophilin b (acute lymphoblastic leukemia); Ig-idiotype (Bcell lymphoma); E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn(glioma); p21ras (bladder cancer); p21ras (biliary cancer); MUC family,HER2/neu, c-erbB-2 (breast cancer); p53, p21ras (cervical carcinoma);p21ras, HER2/neu, c-erbB-2, MUC family, Cripto-1protein, Pim-1 protein(colon carcinoma); Colorectal associated antigen (CRC)-CO17-1A/GA733,APC (colorectal cancer); carcinoembryonic antigen (CEA) (colorectalcancer; choriocarcinoma); cyclophilin b (epithelial cell cancer);HER2/neu, c-erbB-2, ga733 glycoprotein (gastric cancer); α-fetoprotein(hepatocellular cancer); Imp-1, EBNA-1 (Hodgkin's lymphoma); CEA,MAGE-3, NY-ESO-1 (lung cancer); cyclophilin b (lymphoid cell-derivedleukemia); MUC family, p21ras (myeloma); HER2/neu, c-erbB-2 (non-smallcell lung carcinoma); Imp-1, EBNA-1 (nasopharyngeal cancer); MUC family,HER2/neu, c-erbB-2, MAGE-A4, NY-ESO-1 (ovarian cancer); ProstateSpecific Antigen (PSA) and its antigenic epitopes PSA-1, PSA-2, andPSA-3, PSMA, HER2/neu, c-erbB-2, ga733 glycoprotein (prostate cancer);HER2/neu, c-erbB-2 (renal cancer); viral products such as humanpapillomavirus proteins (squamous cell cancers of the cervix andesophagus); NY-ESO-1 (testicular cancer); and HTLV-1 epitopes (T cellleukemia).

Foreign antigens are suitably selected from transplantation antigens,allergens as well as antigens from pathogenic organisms. Transplantationantigens can be derived from donor cells or tissues from, e.g., heart,lung, liver, pancreas, kidney, neural graft components, or from thedonor antigen-presenting cells bearing MHC loaded with self antigen inthe absence of exogenous antigen.

Non-limiting examples of allergens include Fel d 1 (i.e., the felineskin and salivary gland allergen of the domestic cat Felis domesticus,the amino acid sequence of which is disclosed International PublicationWO 91/06571), Der p I, Der p II, Der fI or Der fII (i.e., the majorprotein allergens from the house dust mite dermatophagoides, the aminoacid sequence of which is disclosed in International Publication WO94/24281). Other allergens may be derived, for example from thefollowing: grass, tree and weed (including ragweed) pollens; fungi andmolds; foods such as fish, shellfish, crab, lobster, peanuts, nuts,wheat gluten, eggs and milk; stinging insects such as bee, wasp, andhornet and the chirnomidae (non-biting midges); other insects such asthe housefly, fruitfly, sheep blow fly, screw worm fly, grain weevil,silkworm, honeybee, non-biting midge larvae, bee moth larvae, mealworm,cockroach and larvae of Tenibrio molitor beetle; spiders and mites,including the house dust mite; allergens found in the dander, urine,saliva, blood or other bodily fluid of mammals such as cat, dog, cow,pig, sheep, horse, rabbit, rat, guinea pig, mouse and gerbil; airborneparticulates in general; latex; and protein detergent additives.

Exemplary pathogenic organisms include, but are not limited to, viruses,bacteria, fungi parasites, algae and protozoa and amoebae. Illustrativeviruses include viruses responsible for diseases including, but notlimited to, measles, mumps, rubella, poliomyelitis, hepatitis A, B(e.g., GenBank Accession No. E02707), and C (e.g., GenBank Accession No.E06890), as well as other hepatitis viruses, influenza, adenovirus(e.g., types 4 and 7), rabies (e.g., GenBank Accession No. M34678),yellow fever, Epstein-Barr virus and other herpesviruses such aspapillomavirus, Ebola virus, influenza virus, Japanese encephalitis(e.g., GenBank Accession No. E07883), dengue (e.g., GenBank AccessionNo. M24444), hantavirus, Sendai virus, respiratory syncytial virus,othromyxoviruses, vesicular stomatitis virus, visna virus,cytomegalovirus and human immunodeficiency virus (HIV) (e.g., GenBankAccession No. U18552). Any suitable antigen derived from such virusesare useful in the practice of the present invention. For example,illustrative retroviral antigens derived from HIV include, but are notlimited to, antigens such as gene products of the gag, pol, and envgenes, the Nef protein, reverse transcriptase, and other HIV components.Illustrative examples of hepatitis viral antigens include, but are notlimited to, antigens such as the S, M, and L proteins of hepatitis Bvirus, the pre-S antigen of hepatitis B virus, and other hepatitis,e.g., hepatitis A, B, and C, viral components such as hepatitis C viralRNA. Illustrative examples of influenza viral antigens include, but arenot limited to, antigens such as hemagglutinin and neuraminidase andother influenza viral components. Illustrative examples of measles viralantigens include, but are not limited to, antigens such as the measlesvirus fusion protein and other measles virus components. Illustrativeexamples of rubella viral antigens include, but are not limited to,antigens such as proteins E1 and E2 and other rubella virus components;rotaviral antigens such as VP7sc and other rotaviral components.Illustrative examples of cytomegaloviral antigens include, but are notlimited to, antigens such as envelope glycoprotein B and othercytomegaloviral antigen components. Non-limiting examples of respiratorysyncytial viral antigens include antigens such as the RSV fusionprotein, the M2 protein and other respiratory syncytial viral antigencomponents. Illustrative examples of herpes simplex viral antigensinclude, but are not limited to, antigens such as immediate earlyproteins, glycoprotein D, and other herpes simplex viral antigencomponents. Non-limiting examples of varicella zoster viral antigensinclude antigens such as 9PI, gpII, and other varicella zoster viralantigen components. Non-limiting examples of Japanese encephalitis viralantigens include antigens such as proteins E, M-E, M-E-NS 1, NS 1, NS1-NS2A, 80% E, and other Japanese encephalitis viral antigen components.Representative examples of rabies viral antigens include, but are notlimited to, antigens such as rabies glycoprotein, rabies nucleoproteinand other rabies viral antigen components. Illustrative examples ofpapillomavirus antigens include, but are not limited to, the L1 and L2capsid proteins as well as the E6/E7 antigens associated with cervicalcancers, See Fundamental Virology, Second Edition, eds. Fields, B. N.and Knipe, D. M., 1991, Raven Press, New York, for additional examplesof viral antigens.

Illustrative examples of fungi include Acremonium spp., Aspergillusspp., Basidiobolus spp., Bipolaris spp., Blastomyces dermatidis, Candidaspp., Cladophialophora carrionii, Coccoidiodes immitis, Conidiobolusspp., Cryptococcus spp., Curvularia spp., Epidermophyton spp., Exophialajeanselmei, Exserohilum spp., Fonsecaea compacta, Fonsecaea pedrosoi,Fusarium oxysporum, Fusarium solani, Geotrichum candidum, Histoplasmacapsulatum var. capsulatum, Histoplasma capsulatum var. duboisii,Hortaea werneckii, Lacazia loboi, Lasiodiplodia theobromas,Leptosphaeria senegalensis, Madurella grisea, Madurella mycetomatis,Malassezia furfur, Microsporum spp., Neotestudina rosatii, Onychocolacanadensis, Paracoccidioides brasiliensis, Phialophora verrucosa,Piedraia hortae, Piedra iahortae, Pityriasis versicolor, Pseudallesheriaboydii, Pyrenochaeta romeroi, Rhizopus arrhizus, Scopulariopsisbrevicaulis, Scytalidium dimidiatum, Sporothrix schenckii, Trichophytonspp., Trichosporon spp., Zygomcete fungi, Absidia corymbifera,Rhizomucor pusillus and Rhizopus arrhizus. Thus, representative fungalantigens that can be used in the compositions and methods of the presentinvention include, but are not limited to, candida fungal antigencomponents; histoplasma fungal antigens such as heat shock protein 60(HSP60) and other histoplasma fungal antigen components; cryptococcalfungal antigens such as capsular polysaccharides and other cryptococcalfungal antigen components; coccidiodes fungal antigens such as spheruleantigens and other coccidiodes fungal antigen components; and tineafungal antigens such as trichophytin and other coccidiodes fungalantigen components.

Illustrative examples of bacteria include bacteria that are responsiblefor diseases including, but not restricted to, diphtheria (e.g.,Corynebacterium diphtheria), pertussis (e.g., Bordetella pertussis,GenBank Accession No. M35274), tetanus (e.g., Clostridium tetani,GenBank Accession No. M64353), tuberculosis (e.g., Mycobacteriumtuberculosis), bacterial pneumonias (e.g., Haemophilus influenzae.),cholera (e.g., Vibrio cholerae), anthrax (e.g., Bacillus anthracis),typhoid, plague, shigellosis (e.g., Shigella dysenteriae), botulism(e.g., Clostridium botulinum), salmonellosis (e.g., GenBank AccessionNo. L03833), peptic ulcers (e.g., Helicobacter pylori), Legionnaire'sDisease, Lyme disease (e.g., GenBank Accession No. U59487). Otherpathogenic bacteria include Escherichia coli, Clostridium perfringens,Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcuspyogenes. Thus, bacterial antigens which can be used in the compositionsand methods of the invention include, but are not limited to: pertussisbacterial antigens such as pertussis toxin, filamentous hemagglutinin,pertactin, FM2, FIM3, adenylate cyclase and other pertussis bacterialantigen components; diphtheria bacterial antigens such as diphtheriatoxin or toxoid and other diphtheria bacterial antigen components;tetanus bacterial antigens such as tetanus toxin or toxoid and othertetanus bacterial antigen components, streptococcal bacterial antigenssuch as M proteins and other streptococcal bacterial antigen components;gram-negative bacilli bacterial antigens such as lipopolysaccharides andother gram-negative bacterial antigen components; Mycobacteriumtuberculosis bacterial antigens such as mycolic acid, heat shock protein65 (HSP65), the 30 kDa major secreted protein, antigen 85 A and othermycobacterial antigen components; Helicobacter pylori bacterial antigencomponents, pneumococcal bacterial antigens such as pneumolysin,pneumococcal capsular polysaccharides and other pnermiococcal bacterialantigen components; Haemophilus influenza bacterial antigens such ascapsular polysaccharides and other Haemophilus influenza bacterialantigen components; anthrax bacterial antigens such as anthraxprotective antigen and other anthrax bacterial antigen components;rickettsiae bacterial antigens such as rompA and other rickettsiaebacterial antigen component. Also included with the bacterial antigensdescribed herein are any other bacterial, mycobacterial, mycoplasmal,rickettsial, or chlamydial antigens.

Illustrative examples of protozoa include protozoa that are responsiblefor diseases including, but not limited to, malaria (e.g., GenBankAccession No. X53832), hookworm, onchocerciasis (e.g., GenBank AccessionNo. M27807), schistosomiasis (e.g., GenBank Accession No. LOS 198),toxoplasmosis, trypanosomiasis, leishmaniasis, giardiasis (GenBankAccession No. M33641), amoebiasis, filariasis (e.g., GenBank AccessionNo. J03266), borreliosis, and trichinosis. Thus, protozoal antigenswhich can be used in the compositions and methods of the inventioninclude, but are not limited to: plasmodium falciparum antigens such asmerozoite surface antigens, sporozoite surface antigens,circumsporozoite antigens, gametocyte/gamete surface antigens,blood-stage antigen pf 155/RESA and other plasmodial antigen components;toxoplasma antigens such as SAG-1, p30 and other toxoplasmal antigencomponents; schistosomae antigens such as glutathione-S-transferase,paramyosin, and other schistosomal antigen components; leishmania majorand other leishmaniae antigens such as gp63, lipophosphoglycan and itsassociated protein and other leishmanial antigen components; andtrypanosoma cruzi antigens such as the 75-77 kDa antigen, the 56 kDaantigen and other trypanosomal antigen components.

The present invention also contemplates toxin components as antigens.Illustrative examples of toxins include, but are not restricted to,staphylococcal enterotoxins, toxic shock syndrome toxin; retroviralantigens (e.g., antigens derived from HIV), streptococcal antigens,staphylococcal enterotoxin-A (SEA), staphylococcal enterotoxin-B (SEB),staphylococcal enterotoxin₁₋₃ (SE₁₋₃), staphylococcal enterotoxin-D(SED), staphylococcal enterotoxin-E (SEE) as well as toxins derived frommycoplasma, mycobacterium, and herpes viruses.

In specific embodiments, the antigen is delivered to antigen-presentingcells. Such antigen-presenting cells include professional or facultativeantigen-presenting cells. Professional antigen-presenting cells functionphysiologically to present antigen in a form that is recognized byspecific T cell receptors so as to stimulate or energies a T lymphocyteor B lymphocyte mediated immune response. Professionalantigen-presenting cells not only process and present antigens in thecontext of the major histocompatability complex (MHC), but also possessthe additional immunoregulatory molecules required to complete T cellactivation or induce a tolerogenic response. Professionalantigen-presenting cells include, but are not limited to, macrophages,monocytes, B lymphocytes, cells of myeloid lineage, includingmonocytic-granulocytic-DC precursors, marginal zone Kupffer cells,microglia, T cells, Langerhans cells and dendritic cells includinginterdigitating dendritic cells and follicular dendritic cells.Non-professional or facultative antigen-presenting cells typically lackone or more of the immunoregulatory molecules required to complete Tlymphocyte activation or energy. Examples of non-professional orfacultative antigen-presenting cells include, but are not limited to,activated T lymphocytes, eosinophils, keratinocytes, astrocytes,follicular cells, microglial cells, thymic cortical cells, endothelialcells, Schwann cells, retinal pigment epithelial cells, myoblasts,vascular smooth muscle cells, chondrocytes, enterocytes, thymocytes,kidney tubule cells and fibroblasts. In some embodiments, theantigen-presenting cell is selected from monocytes, macrophages, Blymphocytes, cells of myeloid lineage, dendritic cells or Langerhanscells. In certain advantageous embodiments, the antigen-presenting cellexpresses CD11c and includes a dendritic cell or Langerhans cell. Insome embodiments the antigen-presenting cell stimulates an immuneresponse. In other embodiments, the antigen-presenting cell induces atolerogenic response.

The delivery of exogenous antigen to an antigen-presenting cell can beenhanced by methods known to practitioners in the art. For example,several different strategies have been developed for delivery ofexogenous antigen to the endogenous processing pathway ofantigen-presenting cells, especially dendritic cells. These methodsinclude insertion of antigen into pH-sensitive liposomes (Zhou andHuang, 1994, Immunomethods, 4:229-235), osmotic lysis of pinosomes afterpinocytic uptake of soluble antigen (Moore et al., 1988, Cell,54:777-785), coupling of antigens to potent adjuvants (Aichele et al.,1990, J. Exp. Med., 171: 1815-1820; Gao et al., 1991, J. Immunol., 147:3268-3273; Schulz et al., 1991, Proc. Natl. Acad. Sci. USA, 88: 991-993;Kuzu et al., 1993, Euro. J. Immunol., 23: 1397-1400; and Jondal et al.,1996, Immunity 5: 295-302) and apoptotic cell delivery of antigen(Albert et al. 1998, Nature 392:86-89; Albert et al. 1998, Nature Med.4:1321-1324; and in International Publications WO 99/42564 and WO01/85207). Recombinant bacteria (e.g., E. coli) or transfected hostmammalian cells may be pulsed onto dendritic cells (as particulateantigen, or apoptotic bodies respectively) for antigen delivery.Recombinant chimeric virus-like particles (VLPs) have also been used asvehicles for delivery of exogenous heterologous antigen to the MHC classI processing pathway of a dendritic cell line (Bachmann et al., 1996,Eur. J. Immunol., 26(11): 2595-2600).

Alternatively, or in addition, an antigen may be linked to, or otherwiseassociated with, a cytolysin to enhance the transfer of the antigen intothe cytosol of an antigen-presenting cell of the invention for deliveryto the MHC class I pathway. Exemplary cytolysins include saponincompounds such as saponin-containing Immune Stimulating Complexes(ISCOMs) (see, e.g., Cox and Coulter, 1997, Vaccine 15(3): 248-256 andU.S. Pat. No. 6,352,697), phospholipases (see, e.g., Camilli et al.,1991, J. Exp. Med. 173: 751-754), pore-forming toxins (e.g., anα-toxin), natural cytolysins of gram-positive bacteria, such aslisteriolysin O (LLO, e.g., Mengaud et al., 1988, Infect. Immun. 56:766-772 and Portnoy et al., 1992, Infect. Immun. 60: 2710-2717),streptolysin O (SLO, e.g., Palmer et al., 1998, Biochemistry 37(8):2378-2383) and perfringolysin O (PFO, e.g., Rossjohn et al., Cell 89(5):685-692). Where the antigen-presenting cell is phagosomal, acidactivated cytolysins may be advantageously used. For example,listeriolysin exhibits greater pore-forming ability at mildly acidic pH(the pH conditions within the phagosome), thereby facilitating deliveryof vacuole (including phagosome and endosome) contents to the cytoplasm(see, e.g., Portnoy et al., Infect. Immun. 1992, 60: 2710-2717).

The cytolysin may be provided together with a pre-selected antigen inthe form of a single composition or may be provided as a separatecomposition, for contacting the antigen-presenting cells. In oneembodiment, the cytolysin is fused or otherwise linked to the antigen,wherein the fusion or linkage permits the delivery of the antigen to thecytosol of the target cell. In another embodiment, the cytolysin andantigen are provided in the form of a delivery vehicle such as, but notlimited to, a liposome or a microbial delivery vehicle selected fromvirus, bacterium, or yeast. Suitably, when the delivery vehicle is amicrobial delivery vehicle, the delivery vehicle is non-virulent. In apreferred embodiment of this type, the delivery vehicle is anon-virulent bacterium, as for example described by Portnoy et al. inU.S. Pat. No. 6,287,556, comprising a first polynucleotide encoding anon-secreted functional cytolysin operably linked to a regulatorypolynucleotide which expresses the cytolysin in the bacterium, and asecond polynucleotide encoding one or more pre-selected antigens.Non-secreted cytolysins may be provided by various mechanisms, e.g.,absence of a functional signal sequence, a secretion incompetentmicrobe, such as microbes having genetic lesions (e.g., a functionalsignal sequence mutation), or poisoned microbes, etc. A wide variety ofnonvirulent, non-pathogenic bacteria may be used; preferred microbes arerelatively well characterized strains, particularly laboratory strainsof E. coli, such as MC4100, MC1061, DH5a, etc. Other bacteria that canbe engineered for the invention include well-characterized, nonvirulent,non-pathogenic strains of Listeria monocytogenes, Shigella flexneri,mycobacterium, Salmonella, Bacillus subtilis, etc. In a particularembodiment, the bacteria are attenuated to be non-replicative,non-integrative into the host cell genome, and/or non-motile inter- orintra-cellularly.

The delivery vehicles described above can be used to deliver one or moreantigens to virtually any antigen-presenting cell capable of endocytosisof the subject vehicle, including phagocytic and non-phagocyticantigen-presenting cells. In embodiments when the delivery vehicle is amicrobe, the subject methods generally require microbial uptake by thetarget cell and subsequent lysis within the antigen-presenting cellvacuole (including phagosomes and endosomes).

In other embodiments, the antigen is produced inside theantigen-presenting cell by introduction of a suitable expression vectoras for example described above. The antigen-encoding portion of theexpression vector may comprise a naturally-occurring sequence or avariant thereof, which has been engineered using recombinant techniques.In one example of a variant, the codon composition of anantigen-encoding polynucleotide is modified to permit enhancedexpression of the antigen in a target cell or tissue of choice usingmethods as set forth in detail in International Publications WO 99/02694and WO 00/42215. Briefly, these methods are based on the observationthat translational efficiencies of different codons vary betweendifferent cells or tissues and that these differences can be exploited,together with codon composition of a gene, to regulate expression of aprotein in a particular cell or tissue type. Thus, for the constructionof codon-optimized polynucleotides, at least one existing codon of aparent polynucleotide is replaced with a synonymous codon that has ahigher translational efficiency in a target cell or tissue than theexisting codon it replaces. Although it is preferable to replace all theexisting codons of a parent nucleic acid molecule with synonymous codonswhich have that higher translational efficiency, this is not necessarybecause increased expression can be accomplished even with partialreplacement. Suitably, the replacement step affects 5, 10, 15, 20, 25,30%, more preferably 35, 40, 50, 60, 70% or more of the existing codonsof a parent polynucleotide.

The expression vector for introduction into the antigen-presenting cellwill be compatible therewith such that the antigen-encodingpolynucleotide is expressible by the cell. For example, expressionvectors of this type can be derived from viral DNA sequences including,but not limited to, adenovirus, adeno-associated viruses, herpes-simplexviruses and retroviruses such as B, C, and D retroviruses as well asspumaviruses and modified lentiviruses. Suitable expression vectors fortransfection of animal cells are described, for example, by Wu and Ataai(2000, Curr. Opin. Biotechnol. 11(2):205-208), Vigna and Naldini (2000,J. Gene Med. 2(5):308-316), Kay, et al. (2001, Nat. Med. 7(1):33-40),Athanasopoulos, et al. (2000, Int. J. Mol. Med. 6(4):363-375) andWalther and Stein (2000, Drugs 60(2):249-271).

Experimental Results

Preliminary data in a mouse model, using a virus as a delivery vehicleto the skin using an embodiment of the patches 100 described aboveelicited significantly higher cytotoxic T-lymphocyte responses thanconventional intradermal injection of the same antigenic preparation,with the same payload.

Accordingly, the above described examples provide a device for thedelivery of a bioactive material (agent) or other stimulus to aninternal site in the body, comprising a plurality (number) ofprojections that can penetrate a body surface so as to deliver thebioactive material or stimulus to the required site.

In one example, the number of projections is selected to be at least500, to thereby induce a biological response. Typically the exact numberof projections is determined in accordance with the above describedequations, thereby maximizing the chance of delivery of material orstimulus to the target.

The delivery end portion of the projection may also be dimensioned so asto be capable of insertion into targeted cells to deliver the bioactivematerial or stimulus without appreciable damage to the targeted cells orspecific sites therein. Thus, the dimensions of the delivery end portionof the microprojections, including the length or diameter, can beselected which enables delivery of the agent or stimulus to targetedcells and internal components within cells.

The nanoneedles are typically solid (non-hollow) in cross-section. Thisleads to a number of technical advantages which include: reliabledelivery of bioactive material or stimulus; ease and cost ofmanufacturing, and increased strength.

Variations

A number of variations and options for use with the above describeddevices will now be described.

Herein, the terms “projection”, “micro-nanoprojection”, “nanoneedle”,“nanoprojection”, “needle”, “rod”, etc., are used interchangeably todescribe the solid projections.

In cases where a material or agent is to be transported, projections maybe coated on the outside of the nanoneedles. This provides a higherprobability of delivering the coating to the depth of interest comparedto microparticle delivery from a gene gun and thus is more efficient.

A further feature is that the nanoneedles may be used for delivery notonly through the skin but through other body surfaces, including mucosalsurfaces, to cellular sites below the outer layer or layers of suchsurfaces. The term “internal site”, as used herein, is to be understoodas indicating a site below the outer layer(s) of skin and other tissuesfor which the devices of the present invention are to be used.

Furthermore, these nanoneedles may be used to deliver stimuli to cellsor cell components other than those resulting from the administration ofbioactive agents such as drugs and antigenic materials for example. Merepenetration of cellular sites with nanoneedles may be sufficient toinduce a beneficial response, as indicated hereinafter.

The device is suitable for intracellular delivery. The device issuitable for delivery to specific organelles within cells. Examples oforganelles to which the device can be applied include a cell nucleus,endoplasmic reticulum, ribosome, or lysosome for example.

In one embodiment the device is provided having a needle supportsection, that is to say the projections comprise a suitable supportsection, of sufficient length to reach the desired site and a (needle)delivery end section having a length no greater than 20 microns and amaximum width no greater than 5 microns, preferably no greater than 2microns.

In one example, the maximum width of the delivery end section is nogreater than 1000 nm, even more preferably the maximum width of thedelivery end section is no greater than 500 nm.

In one embodiment, the device can be used for delivery intradermally.This device may have a needle support section, that is to say theprojections comprise a suitable support section, of length at least 10microns and a (needle) delivery end section having a length no greaterthan 20 microns and a maximum width no greater than 5 microns,preferably no greater than 2 microns.

The maximum width of the delivery end section is usually no greater than1000 nm, even more preferably the maximum width of the delivery endsection is no greater than 500 nm.

In a further embodiment, the device is for mucosal delivery. This devicemay have a needle support section, that is to say the projectionscomprise a suitable support section, of sufficient length to reach thedesired site, such as of length at least 100 microns and a (needle)delivery end section having a length no greater than 20 microns and amaximum width no greater than 5 microns, preferably no greater than 2microns.

In one embodiment, the device of the invention is for delivery to lungor other internal organ or tissue. In a further embodiment, the deviceis for in-vitro delivery to tissue, cell cultures, cell lines, organs,artificial tissues and tissue engineered products. This device typicallyhas a needle support section, that is to say the projections comprise asuitable support section, of length at least 5 microns and a needledelivery end section having a length no greater than 20 microns and amaximum width no greater than 5 microns, preferably no greater than 2microns.

In one embodiment, the device comprises projections in which the(needle) delivery end section and support length, that is to say the“needle support section”, is coated with a bioactive material across thewhole or part of its length. The (needle) delivery end section andsupport length may be coated on selective areas thereof. This may dependupon the bioactive material being used or the target selected forexample.

In a further embodiment, a bioactive material is releasably incorporatedinto the material of which the needle, or projection, is composed. All,or part of the projection may be constructed of a biocompatible,biodegradable polymer (such as Poly Lactic Acid (PLA), PolyGlycolic Acid(PGA) or PGLA or Poly Glucleic Acid), which is formulated with thebioactive material of choice. The projections may then be inserted intothe appropriate target site and, as they dissolve, the bioactivematerial will enter the organelle(s)/cells.

In one example, at least the delivery end section of the needle iscomposed of a biodegradable material.

In an alternative embodiment of the invention, a device is also providedin which the needle has no bioactive material on or within it. Thetargeted cell or organelle is perturbed/stimulated by the physicaldisruption caused by the (delivery end of the) nanoneedle (projection).This physical stimulus may, for example, be coupled with electricstimulus as a form of specific nanoelectroporation of particularorganelles or the cell.

The bioactive material or stimulus delivered by the device of theinvention may be any suitable material or stimulus which gives thedesired effect at the target site.

Examples of bioactive materials, which are not intended to be limitingwith respect to the invention include polynucleotides and nucleic acidor protein molecules, antigens, allergens, adjuvants, molecules,elements or compounds. In addition, the device may be coated withmaterials such as biosensors, nanosensors or MEMS.

In one aspect, the device is provided in the form of a patch containinga plurality of needles (projections) for application to a body surface.A multiplicity of projections can allow multiple cells and organelles tobe targeted and provided with a bioactive material or stimulus at thesame time. The patch may be of any suitable shape, such as square orround for example. The overall number of projections per patch dependsupon the particular application in which the device is to be used.Preferably, the patch has at least 10 needles per mm, and morepreferably at least 100 needles per mm². Considerations and specificexamples of such a patch are provided in more detail below.

Examples of specific manufacturing steps used to fabricate the deviceare described in greater detail below. In one preferred aspect, thedevice of the invention is constructed from biocompatible materials suchas Titanium, Gold, Silver or Silicon, for example. This may be theentire device, or alternatively it may only be the projections or thedelivery end section of the projections which are made from thebiocompatible materials.

One manufacturing method for the device utilizes the Deep Reactive IonEtching (DRIE) of the patterns direct from silicon wafers, see theconstruction section below.

Another manufacturing method for the device utilizes manufacturing froma male template constructed with X-ray lithography, electrodepositionand molding (LIGA). The templates are then multiply inserted into a softpolymer to produce a plurality of masks. The masks are then vacuumdeposited/sputtered with the material of choice for the nanoprojections,such as titanium, gold, silver, or tungsten. Magnetron sputtering mayalso be applied, see the construction section below.

An alternative means for producing masks is with 2 photonStereolithography, a technique which is known in the art and isdescribed in more detail below.

In one embodiment, the device is constructed of silicon.

The device may be for a single use or may be used and then recoated withthe same or a different bioactive material or other stimulus, forexample.

In one embodiment, the device comprises projections which are ofdiffering lengths and/or diameters (or thicknesses depending on theshape of the projections) to allow targeting of different targets withinthe same use of the device.

An example of the practical application of such a device is explained infurther detail below (see section 6.2.4).

Also provided throughout the specification are numerous uses of thedevice, which has many useful medical applications in the treatment of anumber of diseases.

Further Examples

Further examples will now be described in more detail. For the purposeof these examples, the explanation will focus on the targeting of cells,cell organelles or cell nuclei. However it will be appreciated that thetechniques may be used to deliver material/stimulus to any suitablespecific target.

1. Specific Targets for Delivery

In one example, the target of interest is within the cells. The positionand shape of key organelles within the cells are shown in FIG. 15. Alleucaryotic cells have the same basic set of membrane enclosedorganelles. The number and volume of the key organelles varies with celltype. As to the targeting of specific organelles, there is a probabilityattached to each event on the basis of volume. Consider the non-specifictargeting of the cell (i.e., at the correct depth, but without theprecise targeting with the aid of imaging techniques). The probabilityof targeting the nucleus, for example, in a cell is given by the volumeof the nucleus vs. the remainder of the cell.

In Table 1 below, the scale of organelles and their mass fraction andnumber are listed. The primary data for this summary is from “Genes VI”by Benjamin Lewin and “The Molecular Biology of the Cell” Alberts etal., 4th Ed. This information pertains to the Liver Cell. Also listed inTable 1 are example applications and that may be induced or enhanced asa result of targeting these organelles.

Working in increasing scale, the starting point is the cell membrane,which is ˜10 nm thick. In piercing these membranes with a minimaldisruption to the viability of the cell, a range of drugs, vaccines andother compounds can be delivered to cells. Specifically, the EndoplasmicReticulum (a convoluted envelope, 75 nm in thickness) may be targetedwithin RNA to transfect cells in the areas of Vaccination or GeneTherapy. Lysosomes, which are 200-500 nm in size can be targeted forrelease of enzymes to induce autolysis (cell death). For an effectivecellular response (MHC Class I) with DNA vaccination and gene therapy,it is important that the DNA material is delivered intact to the cellnucleus.

Most of these organelles are of the submicron scale.

TABLE 1 The scale, number and potential targets of key organelles withincells. (http://niko.unl.edu/bs101/notes/sizes.html and “MolecularBiology of the Cell” 4″′ Ed, Alberts et al.) Mass/Volume Number Utilityas Scale Organelle Fraction per cell target Application ~0.1 nm HydrogenAtom ~0.8 nm Amino Acid ~2 nm DNA Alpha helix ~2% (diameter) ~2 nm mRNA~2% ~2,500 ~2 nm tRNA ~3% ~160,000 ~2 nm RNA ~21%  ~4 nm GlobularProtein ~10 nm Cell membranes ~10%  Use nanoneedles to pierce To enhancedrug/ these membranes with vaccine delivery minimal disruption to ofcells through the cell. perfusion ~11 nm Ribosome ~9% ~25 nm Microtubulediameter ~75 nm Endoplasmic reticulum ~15%  Target with RNA, mRNA,Vaccination, (envelope (smooth, rough, Golgi) iRNA for up regulating orgene therapy thickness) interfering the production for cancerous ofproteins cells. ~100 nm Large Virus ~200-500 nm Lysosomes ~1% Pierce thelysosomes to Inducing cell release enzymes and death in inducesautolysis (self cancerous cells. digestion of the cell). ~3 μm ×Mitochondrion ~22%  200 nm ~3-6 μm Nucleus ~6% (Liver) 1 Target for DNAdelivery to DNA vaccination 16% (LC)   the cell directly and/or or genetherapy. disturb the double membrane. ~10-30 μm Cell Langerhans cellsare ~10 (diameter) μm in diameter.

2. Routes of Delivery to Cells

The type and scale of organelles within cells has been presented. Nowthe location of these cells within the tissue is identified. Theselected tissue routes are.

-   -   Intradermal    -   Mucosal    -   Lung    -   Internal tissues    -   In-vitro sites.

2.1 Intradermal Delivery

The skin is one convenient route for drug and vaccine delivery. Aschematic of the skin is shown in FIG. 16. A full description of theanatomy of the skin is given in textbooks. A closer view of a histologysection of skin is shown in FIG. 17 in a photomicrograph. Respectivethicknesses of layers vary between species and sites.

A key barrier to many drugs and vaccines is the Stratum Corneum (SC). Inhumans, this barrier is 10-20 μm thick with large variations fromsite-to-site with different ages and sexes. Below the SC is the ViableEpidermis (VE), which is typically 40-60 μm thick in humans. Within theVE are immunologically sensitive Langerhans Cells (LC). FIG. 17 shows astained LC, marked as “L”, residing above the basal layer. The spatialdistribution of LC is illustrated in a 3D distribution of LC in a mouse,FIG. 18 (Kendall M. A. F., Mulholland W. J., Tirlapur U. K., ArbuthnottE. S., and Armitage, M. (2003) “Targeted delivery of micro-particles toepithelial cells for immunotherapy and vaccines: an experimental andprobabilistic study”, The 6th International Conference on CellularEngineering, Sydney, August 20-22).

There are typically about 1000 LC per square mm and they reside justabove the basement membrane of the viable epidermis (i.e., 30-80 μmdeep). Importantly, the depth of LC varies significantly with thebiological variability of the tissue, including rete ridges etc.

One objective in vaccination and gene therapy applications is to targetLC residing 30-80 μm below the skin surface. In order to do this, the SCand cell membranes are to be breached. Furthermore, the organelleswithin the LC are to be targeted to elicit particular responses. Oneexample is in the triggering of MHC I responses, whereby intact DNA isto be delivered to cell the nucleus. Alternatively, mRNA can bedelivered to the Endoplasmic Reticulum or the cytoplasm.

Similarly, in the treatment of skin cancers such as Squamous CellCarcinoma, cancerous cells within the VE may be directly targeted.

In some applications it may be beneficial to target cells deeper intothe dermis. For example, in Basal Cell Carcinoma, the cells to betargeted are deeper in the tissue, approaching hundreds of microns (600μm to 800 μm) (British Journal of Dermatology 149(5) Page 1035-2003).

2.2 Mucosal Delivery

FIG. 19A shows a photomicrograph of a histology section of the mucosaand FIG. 19B shows the structure of the mucosa. The physiology of themucosa is similar to the skin. However there are some notabledifferences:

-   -   the mucosa does not have a stratum corneum    -   the epithelium is 600-800 μm thick. This is considerably deeper        than the epidermis of the skin (the exact thickness varies with        site, age and species).

Therefore, to target organelles within cells at the basement membrane ofthe epithelium, a depth D_(c) of 600-800 μm is required.

2.3 Lung Delivery

The epithelial cells on the lining of the lung are the target forvarious gene therapy approaches. These cells are underneath amucous/liquid lining, which may be 10-100 μm thick. Therefore thislining needs to be overcome. In the example of gene therapy for thetreatment of Cystic Fibrosis, this lining is to be overcome to targetcelial cells on the surface of the epithelium.

2.4 Other Internal Organ Delivery

Cells within other the internal organs can also be targeted for a rangeof applications, such as:

-   -   Internal cancers    -   Liver (e.g., for Malaria treatment)    -   Heart (e.g., for angiogenesis blood vessel formation).

2.5 In-Vitro Delivery

Tissue, cell-lines, tissue culture, excised organs, tissue engineeredconstructs and artificial tissues may also be targeted, in-vitro.Examples include:

-   -   stimulation of cell lines and cell monolayers in culture.    -   stimulation/delivery of growth factors and/or genes (e.g., in        tissue engineering and wound healing).

3. Constructional Features of the Micro-Nanoprojections 3.1 GeneralMicro-Nanoprojection Dimensions

With typical cell and organelle scales described and locations intissues highlighted, the structural terms of the nanoneedlesindividually are described, and then as examples of arrays for targetingcells at different tissue sites.

Nanoneedles are configured to penetrate the cell membrane with minimaldamage, targeting the organelles of interest. The overall dimensions ofthe nanoneedle projection are shown in FIG. 20. The nanoneedle isdivided into two main sections:

3.1.1 The Targeting Section, Length (l)

Referring to FIG. 20, the radius of the tip (r) is to be as small aspracticable with limits set by manufacturing methods and materialconsiderations, where usually the diameter at the distal end of l is d₁.Usually d₁˜2r. The diameter of the upper end of l is defined as d₂. Overthe length l, the effective diameter, tapering from d₁ to d₂ is to beconsiderably less than the diameter of the cell (d_(cell)) or othertarget. It is shown in Table 1 approximately that 10 μm<d_(cell)<30 μm.So, approximately, d₁<<10 μm, preferably <1 μm, ideally in some cases<500 nm. Ideally, the scale of d₁ is to be of the order of the organelleof interest.

For practical engineering and manufacturing and purposes (e.g.,buckling/loading/fracture) it is often preferred that the projectionalong l tapers out to a larger diameter (d₂>d₁) that still is less thanthe diameter of the cell (<d_(cell)). However, along the length of l,the profile may be configured such that the diameter is constant (i.e.,d₁=d₂).

The length of the targeting section (l) is sufficient to ensure that theorganelle of interest is targeted (i.e., l>organelle dimension). Forexample, in targeting the cell nucleus, l>3-6 μm). Ideally, l is evenlonger to account for variation in cell depth location (e.g., as shownby the variation in the Langerhans cell depth Figure A) and increase theprobability of the desired needle-organelle contact. The upper length ofl is determined by the combination of material properties, needle shapeand loading to ensure that the needle does not break under mechanicalloading. Engineering analyses shows this mechanical loading is mostlycompression, with tension and bending moments. Good engineering practiceto ensure the material does not break includes Euler buckling, fracturemechanics, and work-to-failure. In considering a population ofprojections in an array, statistical methods (e.g., Weibull statistics)and other related methods may be applied to ensure a very small fractionof the projection population breaks.

Consider, as an example, the case of compression loading with theprimary mode of projection failure is by buckling. This compressionbuckling criteria set by the Euler Bucking force (P_(cr))

$P_{cr} = \frac{\pi^{2}{EI}}{4l^{2}}$

where (E) is the Young's modulus of the nanoneedle material, I is themoment of inertia (I=

$\frac{\pi \; d^{4}}{64}$

for a cylinder). Therefore the material properties of the nanoneedle andshape determine the maximum force permitted for buckling (P_(cr)). Goodengineering practice dictates that P_(cr) must be less than theinsertion force of the needle, to ensure it does not break.

3.1.2 Support Section of the Micro-Nanoprojection (a)

Referring again to FIG. 20, the support section of themicro-nanoprojection (a) is sufficient to bring the targeting section ofthe cell (l) into contact with the cells/organelles of interest. Thediameter of the support section along the length a tapers at the distalend from d₂ to d₃ at the base, where usually engineering and materialconsiderations result in d₃>d₂ to ensure it does not buckle or break byanother mode of failure. In some cases, the diameter along the length ofa may be constant (i.e., d₃=d₂). FIG. 20 shows that a, l and the overalllength of the projection (L) are related by:

L=a+l

In section 2 the depths of cells for various applications inintradermal, mucosal, lung and internal organ delivery are outlined. Therationale and approximate values of a for targeting these individualsites are presented as examples.

In epidermal delivery, a is the sum of the desired depth in the tissueand an allowance above the target (for instance for tissue surfacecurvature). This dimension in epidermal delivery is usually <200 μm inlength, and preferably <100 μm in length-depending on the tissue speciesand site. In the targeting of dermal cells, this depth is even greaterwith a<1000 μm, and preferably <600 μm. In another example, thetargeting of basal cells in the epithelium of the mucosa, an a of600-800 μm is required. In lung delivery, the local bather of the mucouslining to the cells is 50-100 μm thus a would be of the order of 100 μmin this case.

3.2 Specific Applications 3.2.1 Example 1

Targeting the nuclei of LC in the viable epidermis, 40-50 μm deep in thetissue. The force to insert the needle is estimated with the UnifiedPenetration Model (proposed by Dehn J: A unified theory of penetration.International Journal of Impact Engineering, 5:239-248.1987).

${F_{insert} \approx {3A\; \sigma_{y}}} = {3\frac{\pi}{4}d^{2}\sigma_{y}}$

where σ_(y) is the yield stress of the tissue.

Assume, in the highest loading case, the upper value of the yield stressof the SC of 20 MPa applies to all the skin (from Wildnauer R H,Bothwell J W, Douglas A B: Stratum corneum properties I. influence ofrelative humidity on normal and extracted stratum corneum. Journal ofInvestigative Dermatology, 56(1):72-78. 1971). Note, this estimate ofinsertion force is a factor of 10 greater than calculations inferredfrom measurements with a probe in the skin (unpublished results, byCrowley (2003, 4^(th) Year Project, Engineering Science, University ofOxford).

By setting F_(insert)=P_(cr) we arrive at

$l = ( \frac{\pi \; {Ed}^{2}}{192\sigma_{y}} )^{0.5}$

which approximates the Euler Buckling relationship between the maximalpermissible length of extension for a probe of diameter d, constructedof a material with a Young's modulus (E), penetrating into a tissue witha Yield Stress (σ_(y)).

FIG. 21 plots the cell/organelle targeting section length (l) as afunction of diameter for six materials. Note that these are upper limitcalculations of the loading effects.

Consider the curve for titanium (E=116 GPa) in which a 400 nm diameter(d₁=d₂) cell/organelle targeting section corresponds to a length of 7μm, and a 1 μm diameter corresponds to a maximum length of 14.1 μm. Inthis case, the nucleus of the LC is the organelle of interest with asize of 3-6 μm. Here, we choose a organelle targeting section of d₁=d₂=1μm and a length (l) of 10 μm (symbols corresponding to the diagram ofFIG. 20). The support length (a) is 40 μm, tapering to a base diameter(d₃) of 5 μm. These dimensions are summarized in Table 2.

Alternatively, a nanoneedle constructed of a stiffer material such assilicon (E=189 GPa) results in a smaller cell/organelle targetingsection of d₁=d₂=200 nm over a length (l) of 5 μm. Of course, a brittlematerial such as silicon would also require fracture (and other)analyses that may lead to more conservative (i.e., larger)dimensions—these numbers throughout this section are simplyillustrative.

Utility of an even stiffer material such as Tungsten (E=400 GPa) resultsin the same nanoneedle diameter of d₁=d₂=200 nm extended over a length(l) of >6 μm—or alternatively the possibility of a smaller diameterd₁=d₂<200 nm for a length of 5 μm.

3.2.2 Example 2

Targeting the Endoplasmic Reticulum LC of the Viable Epidermis. Table 1lists the thickness of the Endoplasmic Reticulum envelope to be 75 nm.Choosing titanium, the organelle targeting section is set at 400 nm overa length of 5 μm. As shown in Table 2, the remainder of the componentsare identical to those set out in Example 1.

3.2.3 Example 3

Targeting cell nuclei at a depth of 600 μm in the tissue. Here, thetargeting section is identical to Example 1, whereas the considerablylonger length (a=600 μm) requires a significantly wider base (d₃=50 μm).

3.2.4 Example 4

FIG. 20 shows an axi-symmetric design for the nanoneedle. Alternativeshapes may be used with:

-   -   higher values of mass-moment of inertia (I)    -   more knife-like geometries with sharper leading edges.

One possible example of an alternative shape taking on these features isshown in plan view in FIG. 27.

TABLE 2 The geometry of three nanoneedle examples. r d₁ d₂ l d₃ aExample (nm) (nm) (nm) (μm) (μm) (μm) 1. Nucleus of LC, <1000 1000 100010 5 40 40-50 μm deep 2. Endoplasmic Reticulum <400 400 400 5 5 45 of LC40-50 μm deep 3. Cell nuclei, 600 μm <400 1000 1000 10 50 600 deep.

4. Route for Transfer of Bioactive Material to End Point (Coating)

Consider the generic shape of the micro-nanoprojection (FIG. 20). Thebioactive material is coated to the outer surface of themicro-nanoprojection. The following lists two example protocols on howthis is done, followed by a list of possible coating combinations.

4.1 Coating DNA on Metallic (e.g., Gold or Tungsten)Micro-Nanoprojections

Consider the case of coating eGFP to tungsten micro-nanoprojections.Essentially, published and well established protocols devised forcoating gold microcarriers for biolistic delivery are employed. Examplesof these protocols are detailed in:

PowderJect and Bio-Rad patents and protocols such as:http://plantsciences.montana.edu/wheat-transformation/biolisti.htm); J.E. Biewenga, O. H. Destree, L. H. Schrama, “Plasmid-mediated genetransfer in neurons using the biolistics technique”, J. Neurosci.Methods 71(1997) 67-75; S. Novakovic, M. Knezevic, R. Golouh, B.Jezersek, “Transfection of mammalian cells by the methods of receptormediated gene transfer and particle bombardment”, J. Exp. Clin. CancerRes. 18 (1999) 531-536; H. Wellmann, B. Kaltschmidt, C. Kaltschmidt,“Optimized protocol for biolistic transfection of brain slices anddissociated cultured neurons with a hand-held gene gun,” J. Neurosci.Methods 92 (1999) 55-64; and, John O'Brien, Sarah C. R. Lummis “Animproved method of preparing microcarriers for biolistic transfection”,Brain Research Protocols 10 (2002) 12-15.

In this example, subsequently applied to the ExperimentalExemplification 1 (section 5.3) the protocol of O'Brien and Lummis(2002) is applied.

Alternatively, in coating silicon, protocols developed for thesub-micron patterning of DNA oligonucleotides for “lab-on-a-chip”technology can be applied to coating the projections on the array (H. B.Yin, T. Brown, J. S. Wilkinson, R. W. Eason and T. Melvin (2004)“Submicron patterning of DNA oligonucleotides on silicon” Nucleic AcidsResearch, 2004, Vol. 32, No. 14 el 18). This process is an example oftwo-stage process for the covalent attachment of DNA oligonucleotidesonto crystalline silicon (100) surfaces. In summary, UV light exposureof a hydrogen-terminated silicon (100) surface coated with alkenesfunctionalized with N-hydroxysuccinimide ester groups result in thecovalent attachment of the alkene as a monolayer on the surface.Submicron-scale patterning of surfaces is achieved by illumination withan interference pattern obtained by the transmission of 248 nm excimerlaser light through a phase mask. The N-hydroxysuccinimide ester surfaceact as a template for the subsequent covalent attachment ofaminohexyl-modified DNA oligonucleotides. Oligonucleotide patterns, withfeature sizes of 500 nm, are reliably produced over large areas. Thepatterned surfaces are characterized with atomic force microscopy,scanning electron microscopy, epifluorescence microscopy andellipsometry. Complementary oligonucleotides are hybridized to thesurface-attached oligonucleotides with a density of ˜7×10¹² DNAoligonucleotides per square centimeter (for example).

Some-possible coating combinations include:

-   -   No coating: The cell is perturbed/stimulated by the physical        disruption of the micro-nanoprojection structure. This physical        stimulus can be coupled with electric stimulus as a form of        specific nanoelectroporation of particular organelles or the        cell.    -   Coating entire nanoneedles: The bioactive material can coat the        whole surface of the nanoneedle (over the length L).    -   Selective coating: For certain applications only parts of the        nanoneedle need be coated. For instance, the targeting        section (l) can be selectively coated by only immersing this        part of the needle in the coating media. Alternatively,        different combinations of various coatings can be made on the        one nanoneedle (combinations of DNA and adjuvants, proteins,        cytokines, inhibitors etc.). Or, in the case of nanoneedle        arrays, different coatings may be provided on different        individual micro-nanoprojections.    -   Coating materials: In addition to bioactive materials (such as        DNA, RNA and proteins), the micro-nanoprojections may also be        coated with nanobiosensors (e.g., quantum dots, nanomachines,        MEMS) using a range of coating protocols.    -   Material formulated in degradable micro-nanoprojections: All, or        part of the micro-nanoprojection can be constructed of a        biocompatible, biodegradable polymer (such as Poly Lactic Acid        (PLA), Poly Glucleic Acid (PGA) or PGLA), which is formulated        with the bioactive material of choice. The micro-nanoprojections        may then be inserted and, as they dissolve, the bioactive        material will enter the organelle(s)/cells.

In the cases of bonding of the biomaterials to the surface of themicro-nanoprojections, access to the organelles may be by the following:

-   -   The bioactive material can stay on the surface of the        nanoneedle, but elicit the response within the organelle/cell.    -   The bioactive material bonds can be broken by exposure of        enzymes, proteins or hydration within the cell/organelle.

5. Experimental Exemplification 1

This section outlines the simplest embodiment of the nanoneedle concept:an individual projection, coated on the surface with a bioactivematerial (here DNA coded for GFP to transfection), used to deliver thismaterial to a living cell, in-vitro, where a biological response ismeasured (transfection) and the cell is alive.

5.1 Fabrication of Micro-Nanoprojection

Following the engineering analyses discussed in section 3, Tungsten wasselected as the material for fabricating the individual nanoprojection,largely because of its high stiffness (E=400 GPa) and ease offabrication.

Individual micro-nano projections were fabricated from tungsten rod ofdiameter 280 μm (Source, ADVENT Research Materials Ltd) usingelectropolishing (approximately following the protocol of Cerezo, A.,Larson D. J. & Smith G. D. W. (2001) “Progress in the atomic-scaleanalysis of materials with the three-dimensional atom probe”. MaterialsResearch Society Bulletin, 26(2): 102-107.

The electropolishing set-up used a solution of distilled water with 4%NaOH molar. For the electropolishing tungsten probes with the sharpesttips, the following settings were used:

-   -   3 mm of probe submersed into the solution    -   10 volts A. C. for approx. 42 seconds.

Following optimization, electropolishing was ceased when probe lengthbegan to diminish (detected by visual sighting by naked human eye).

5.2 Example of Individual Electropolished Projections

Recall the dimension parameters correspond to the genericmicro-nanoprojection tip outlined in the schematic of FIG. 20.

FIGS. 22A-B shows Transmission Electron Microscopy (TEM) of anelectropolished Tungsten tip at two different resolutions, with thescale bar in FIG. 22A of 20 μm, and in FIG. 22B of 0.11 μm. FIGS. 22Band 22C show the minimum tip size (d₁) is ˜100 nm (i.e., r˜50 nm). Byusing the electropolishing process, the projection gradually tapers tothe full thickness of the original rod (i.e., d₃=280 μm). In this case,the cell targeting length (l) is approximately 20 μm-defined by wherethe diameter (d₂) remains less than ˜1 μm.

Individual micro-nano projections were glued into hole drilled intoperspex cylindrical holders to allow fitting into microprojectionsystems (see section 5.5).

5.3 Coating of Micro-Nanoprojections with eGFP DNA Plasmid

The tungsten micro-nanoprojections were coated by adapting a protocoldevised for the coating of gold microparticles for biolistic deliverydeveloped by O'Brien and Lummis (2002) “An improved method of preparingmicrocarriers for biolistic transfection”, Brain Research Protocols 10(2002) 12-15. The adapted protocol is as follows:

-   -   (i) Coat tungsten micro-nanoprojection in 50% glycerol/50% water        solution by dipping the wire in the solution for 10 minutes.    -   (ii) Remove the tungsten.    -   (iii) Individual tungsten micro-nanoprojections were placed in a        tube to which was added 50 ml of 1 M spermidine for 5 minutes.    -   (iv) Add 1 μg/ml of 50 ml of DNA Spermidine and gently mix the        two solutions with aspiration with the wire in the solution.    -   (v) Gently, the tube was stirred for a few minutes, and then,        whilst stirring, 1 M CaCl in solution was added in a drop wise        manner.    -   (vi) Gently mix the solution and let DNA precipitate on to the        wire for 30 minutes.    -   (vii) Remove wire from solution and dip it in 100% ethanol.        Repeat this process 3 times.    -   (viii) Let the wire dry before use.    -   (ix) The process was then repeated for the other        micro-nanoprojections.

FIGS. 23A and 23B show images of lengths of tungsten wire (280 μmdiameter) imaged with a fluorescent microscope with uncoated wire inFIG. 23A and DNA coated wire (using the described protocol, above)immersed in a liquid with a propidium iodide stain (red fluorescing dye)in FIG. 23B. As expected there is no red fluorescence with the uncoatedwire, while the strong red fluorescence of the NDA coated tungstenqualitatively shows that the coating was successful.

5.4 Assessment of Delivery into In-Vitro Test Bed

To assess the delivery profile of DNA in the in-vitro equivalent of skintissue, DNA coated projections were inserted into an agar (3%) gelstained with acridine orange (green fluorescing dye). This gel (withoutthe stain) is routinely used for in-vitro testing of biolistic todevices for DNA vaccination and other applications.

The uncoated and coated tungsten rods were inserted and removed by handinto the agar (to a depth >1 mm) for a cycle duration of approximately 1second.

FIGS. 24A-24D show optically sectioned Multi-Photon Microscopy (MPM)images of the agar after insertion of a DNA coated tungsten probe on thesurface in FIG. 24A, at a depth of 13 μm in FIG. 24B, at a depth of (32μm) in FIG. 24C. FIG. 24D shows an optical section at 32 μm of agar gelfollowing insertion of a probe without a DNA coating.

On the surface of the agar, there is very little fluorescence,indicating little DNA. In contrast, at depth sections of 13 μm and 32 μmthe fluorescence in the coated case is significant-indicating that DNAremained intact on the surface of the probe during insertion and thencame off by exposure of the gel and/or by the action of removing theprobe. This fluorescent signal was very strong compared with theuncoated probe control at 32 μm.

5.5 Experiment of DNA Delivery Into a Cell

The described electropolished tungsten micro-nanoprojections coated withGFP plasmid DNA were tested, with the objective of determining whetherthe DNA could be delivered to transfect the cell, without damaging thecell.

The cell line is called A549 which is a human lung carcinoma(epithelial) cell line. Cells were cultured in Dulbecco's modified eaglemedium (DMEM) supplemented with 10% fetal calf serum at 37° C., 5% CO₂.

These cells were mounted on three separate Petri dishes with an opticalslide underneath. The adherent properties of the cells ensured theyremained in contact with the base of the dish.

One of the Petri dishes with cells was set aside as a control. The othertwo Petri dishes were probed with the micro-nanoprojections. In total, 6micro-nanoprojection systems were used.

Probing of the cells was performed with a microinjection system(Eppendorf Femtoject and Injectman NI2 microinjection kit) fitted to aninverted fluorescent microscope (Zeiss Axiovert 25 microscope with stageheater).

The micro-nanoprojection were fitted into the standard microinjectionholder and then moved with course and fine adjustment of a motorizedX-Y-Z axis controller to just contact the cell membrane, beforeretraction of less than 1 μm.

A cycle time of probing was set between limits of 0.1 and 15 seconds,with a traverse distance of 3-5 μm-then the cycle was automaticallyperformed on the system.

After 3 cells, the probe was replaced and refitted with a replacement.In total 10 cells were perturbed with a micro-nanoprojection coated ineGFP plasmid DNA.

All the cell samples were then incubated for 24 hours before viewingwith a wide field fluorescent microscope, with the appropriatefluorescent filters. The observed results show: in the control there isno sign of eGFP transfection, as expected. However, in the cellmicro-nanoprojection cases, we see that 10 cells have transfected. Thuswe have a 100% transfection efficiency and no evidence of cell death.

These data prove the micro-nanoprojections, coated in DNA canindividually target cells, transfecting them and not kill them.

Also, subsequent analysis of the micro-nanoprojections showed theyremained intact, illustrating that unlike biolistic delivery, the“carrier” material is not left behind.

6. Experimental Exemplification 2

With the biological result achieved on a single cell, in-vitro(Experimental exemplification 1). this section outlines a logicalprogression, which is the physical testing of an individual micro-nanoprojection into a representative skin sample for structuralintegrity-thereby testing the engineering analyses outlined in section3.

6.1 Experiment Design

Using the electroporation process described in Embodiment 1, amicro-nanoproprojection was fabricated of Tungsten with a tip radius (r)of <400 nm.

The tissue was freshly excised Balb/C mice ears (age 8-10 weeks), whichwere glued to a metal cylinder holder. The reported stratum corneum andviable epidermis thicknesses of these samples are respectively ˜5 μm and12 μm (Arbuthnott (2003)).

The tissue was indented by fitting the micro-nanoprojection to aNanoindenter (MTS systems, UK), measuring a force displacement curve.FIG. 26 shows two typical sample results of loading-unloading curvesachieved to a depth of ˜50 These results show that themicro-nanoprojection probe penetrated through the epidermis and wellinto the dermis without damage. The difference in magnitude between thecurves is typical of biological variability observed in bio-viscoelastictissue. Importantly, unlike ballistic particle delivery, the depth ofpenetration is not dependent upon this variability-rather the length ofprojection.

Indeed, the projections did not break—the loading experiment was simplystopped at a 50 μm setting on the experimental apparatus. This wasconfirmed by imaging with projections with a microscope after theexperiment. This experiment was repeated 20 times, confirming theengineering analyses of probe structure required to target cells in theskin is applicable and valid.

7. Experimental Exemplification 3: Overall Size Range of IndividualPatch

With the biological and engineering criteria investigated on anindividual projection, the next logical step is to extend the concept toarrays of patches targeting tissue sites. Presented are several patcharrangements, each designed for a particular targeting need of cells,organelles and/or tissue sites.

7.1 General Dimensions and Design of Patch

FIG. 28 shows a schematic of an array of the describedmicro-nanoprojections configured on a patch, with the spacing betweenmicro-nanoprojections defined as (S) and the patch breadth defined as(B). Recall, the basic dimensions and definitions of an individualmicro-nanoprojection are outlined in section 3 and shown schematicallyin FIG. 20; these are referred to again here.

Operation of the patch with the micro-nanoprojections is, for example:

-   -   (a) the slow insertion, by hand (or other means, such as a        spring) of the patch onto the tissue, with the        micro-nanoprojections inserting into the target tissue of        interest.    -   (b) the patch including the micro-nanoprojections are held in        place for a sufficient time for the “event” (biological,        physical or other) to take place. This may be instantaneous, or        in other cases could take days, weeks or months.    -   (c) the patch including the micro-nanoprojections are then        retracted.

These three stages form a cycle, that may be operated by hand orautomated with the aid of suitable mechanical, electrical orelectro-mechanical devices.

Patches may be applied to the tissue site once, or a multitude of timesdepending upon the effect desired.

The case examples presented in 7.2 all center on the targeting ofLangerhans cells or organelles within these cells and the overalldimensions of the individual micro-nanoprojections are configured, usingthe approximate guideline of Example 1 and 2 shown in Table 2. However,as Table 2 also shows, these parameters (r, d₁, d₂, d₃, a) can varysignificantly, depending on the cell/organelle and its position relativeto the tissue surface.

7.2 Case Examples of Patch Configurations

The overall size (B×B) of the patch and spacing (S) between themicro-nanoprojections is determined by the application. Note, in allcases, the patch could be square (B×B), circular, elliptic, or any othersuitable shape. For simplicity, the square dimensions are quotedthroughout. Generally the size is to be less than 15×15 mm (B), with1<S<1000 μm, preferably with 10<S<200 μm.

Note that an alternative for these and other patch examples is forlarger-patches, giving amplified responses and/or which may be easier tohandle having regard to end users/patients/practitioners.

7.2.1 Targeting LC Nuclei

Consider Example 1 from Table 2, which is the targeting of nuclei ofLangerhans cells. Kendall et al. (2003); Kendall M. A. F., Mulholland W.J., Tirlapur U. K., Arbuthnott E. S., and Armitage, M. “Targeteddelivery of micro-particles to epithelial cells for immunotherapy andvaccines: an experimental and probabilistic study”, The 6^(th)International Conference on Cellular Engineering, Sydney, August 20-22),suggest that to trigger cellular responses in DNA vaccination, of theorder of 100 nuclei of LC are effectively transfected in gene gunapplications that lead to the desired cellular (MHC 1) systemicresponse. A probability analysis has been performed to determine theconfiguration of patch required to transfect 100 cells. The probabilityof one micro-nanoprojection making contact with an organelle (or cell)defined over an area and volume is set by:

$P_{contact} = {\frac{V_{probe}}{V_{layer}} \cdot \frac{V_{organelles}}{V_{layer}}}$

In the analysis, it is assumed:

-   -   that there is no cell death.    -   the cell/organelle targeting sections (l) are configured to be        at the correct depth (i.e., the depth of the Langerhans cells),        and, for simplicity, parallel and 1 im in diameter (d₁=d₂=1 μm).    -   the cell/organelle targeting sections (l) are coated in DNA. Of        course, for simplicity of coating, it is possible that most or        all of the micro-nanoprojection structure is coated in DNA.    -   contact of any part of the micro-nanoprojection along the        cell/organelle targeting section with the nucleus is the only        mode that leads to transfection (i.e., cytoplasm delivery,        cross-priming and other modes are ignored).

With a 1 μm diameter cell/organelle targeting section (1), theprobability of contact with a LC nucleus is 0.0131. This probability is1000-1500 times higher than a typical probability of a direct “hit” withthe biolistics approach and microparticles. Furthermore, thetransfection comparison is even more favorable for themicro-nanoprojection patch, given far fewer cells are killed than by thegene gun.

Table 3 is a summary of configurations of patches that may be used. Atone end of the range, a spacing of 100 rods/mm² corresponds to a patchsurface area of 76 mm², with a spacing (S) of 100 nm, a breadth of 8.7mm and a total number of rods of over 7000. Increasing density of rodsto 1000/nm² results in a reduction in patch size to less than 3 mm×3 mm.

TABLE 3 Calculated Patch Configurations for the Targeting of Nuclei ofLC. Area of Spacing Breadth of Total Rod/mm² patch (mm²) (S) (μm) patch(B) (μm) Rods 100 76.4 100 8.7 7639 500 15.3 45 3.9 7639 1000 7.6 32 2.87639

7.2.2 Targeting the LC

In Table 4, this analysis is extended to the configuration of patchrequired to target 100 LC (i.e., anywhere within the complete cell),using micro-nanoprojections with the same geometry as above, with theprobability of the event 0.063. Not surprisingly, this is higher to thanthe probability of targeting the nuclei. Hence, fewer rods are neededand the patch size can be smaller (4×4 mm down to 1.25×1.25 mm).

TABLE 4 Calculated Patch Configurations for the Targeting of AnywhereWithin Complete LC. The Nanoneedle Diameter d₁ Is Assumed to Be 1 μm.Area of Spacing Breadth of Total Rod/mm² patch (mm²) (S) (μm) patch (B)(μm) Rods 100 15.8 100 4.0 1578 500 3.2 45 1.8 1578 1000 1.5 32 1.3 1578

7.2.3 Targeting the Endoplasmic Reticulum of the LC

In Table 5, the analysis is also applied to the targeting of theEndoplasmic Reticulum (Example 2 from Table 2), which may be requiredfor targeted RNA delivery. Using the assumptions from above, with aprobe diameter of 400 nm the probability of a single needle contactingthe Endoplasmic Reticulum is 0.031. In this case, the size of patchranges from 8×8 mm to 2.5×2.5 mm

TABLE 5 Calculated Patch Configurations for the Targeting of theEndoplasmic Reticulum of LC. The Nanoneedle Diameter Is Assumed to be400 nm Area of Spacing Breadth of Total Rod/mm² patch (mm²) (S) (μm)patch (B) (μm) Rods 100 65 100 8.0 6451 500 13 45 3.60 6451 1000 6.5 322.54 6451

7.2.4 A Combination of Micro-Nanoprojection Geometries on a Patch

A patch may also have combinations of micro-nanoprojections, withdifferent geometries. For example the length (L) or indeed the otherparameters in FIG. 20 may be varied throughout the patch, either indefined sequences, clusters and/or randomly (within limits). Withinthis, or separately, the diameter of the projection may be increased onindividual micro-nanoprojections significantly in order to induce celldeath at controlled locations. This, for example, may be used to inducebystander biological responses (e.g.,stimulation/inflammation/activation) to neighboring healthy cells-whichcould have or will be interacting with described, smallermicro-nanoprojections configured for minimal cell damage.

8. Specific Bioactives, e.g., Nucleic Acids

The bioactive or other stimulus is to be coated or part of thenanoneedle array. The choice of bioactive is determined by theapplication and target organelles or cells of interest. This rangeencompasses, but is not restricted to:

-   -   No coating    -   Polynucleotides, DNA (all variants), RNA (all variants),        proteins, antigens, allergens and adjuvants, molecules,        compounds.    -   Biosensor molecules and compounds and materials.    -   Nanosensors (MEMS etc.).    -   Combinations of the above, on a

9. Methods of Production of Device

General requirements of the manufacturing method are:

-   -   a radial resolution of <200 nm;    -   to construct the nanoneedle array on a patch (e.g., FIG. 19) in        a scaleable process for high throughput manufacturing with        minimal human input.    -   Construction to be of a medical grade material (e.g., Gold,        Silver, Titanium, Tungsten or PLA, PGA, PGLA, Silicon)

A range of techniques for micro-nanofabrication methods described intext books, papers and in other literature (e.g., Madou M. J.“Fundamentals of Microfabrication. The science of miniaturization”, CRCPress, 2002; McAllister et al. 2003, PNAS, Nov. 25, 2003, Vol 100,number 4) are applicable to the nanoneedle device here.

Furthermore there are a range of materials and fabrication methods beingdeveloped that also have potential utility as part of amicro-nanoprojection array. Three construction embodiments are shownhere as examples.

In these constructional embodiment example cases, the patch geometry ofmanufacture is summarized in Table 3, with an area of patch ˜76.4 mm², aspacing of 100 μm and the minimum tip size of 1 μm (d₁). A schematic ofthis array on patch is shown in FIG. 29. Of course, the techniques canbe applied to a range of geometries.

9.1 Constructional Embodiment Example 1

As an example of the fabrication of silicon micro-nanoprojections, thefollowing is applied. Deep Reactive Ion Etching (DRIE) is used as aprocess ideal for these high aspect ratio structures, where in oneexample (Oxford Instruments Plasmalab System 100, Modular ICP180 EtchSystem—S12), etch rates of >2.5 μm/min, and sometimes >5 μm/min, arepossible. Full details of etch protocols for this system are availablein the literature, including Oxford Instruments company manuals.

Briefly, chromium was sputter deposited then lithographically patternedas an array of dots onto 3 inch (75 mm) 100 oriented silicon wafers. Thearray of dots had a center-to-center spacing of 100 μm, and in someearlier test cases, 10-20 Similarly, through iteration with theconditions and exploring the extent of undercut, the diameter of thedots 1 μm, and in other cases, 3 μm, 5 μm, and 10 μm. Deep Reactive IonEtching (DRIE, Oxford Instruments, Bristol, UK) was then carried out toobtain the desired by profile by adjusting the etch rate.

The typical instrument conditions included a 20 standard cm³/min (sccm)SF₆ and 15 sccm (O₂) at a pressure of 20 Pa. Power was varied. Theetching process was performed at cryo-cooled conditions, achievingtemperatures of −100 to −150° C. Lower etch rates were used for thetapered sections, whereas higher rates were used for the more parallelsections. Micro-nanoprojection fabrication was finished when thechromium masks became fully undercut and fell off the projection tips.The process is completed in less than 100 minutes, resulting in severalpatch configurations on the wafer.

The patches were then cut from the silicon wafer, with a dedicatedcutter.

To increase the strength of the base of the patch, an additional,thicker material is affixed to the back surface (i.e., the surfacewithout the projections).

This process is repeated to fabricate a large quantity ofmicro-nanoprojection array patches.

A variation of the described DRIE method may also be applied to othermaterials, including Silicon Carbide (SiC).

9.2 Constructional Embodiment Example 2

This constructional embodiment example applies to a broader range ofmicro-nanoprojections materials than those described in theconstructional embodiment Example 1. These materials include Metals,polymers, silicon and oxides/carbides.

As an example, this case illustrates a method of fabricating tungstenmicro-nanoprojection arrays on a patch. Three of the steps in thisproduction process are discussed.

Step 1. Construction of a Template (Male)

To construct a male template with the profile of themicro-nanoprojections, LIGA is used (a German acronym for X-raylithography, electrodeposition and molding). This template will serve inthe production of several molds in a soft polymer. LIGA, which utilizesa Synchroton, is ideal for creating the template because it has a veryhigh resolution (<20 nm), can fabricate in metals, and will easilyfabricate a patch (maximum component size is 3.4 inches in the Axsuntechnologies system). Fabrication protocols to construct the templateare detailed in Chapter 6 of Madou M. J. “Fundamentals ofMicrofabrication. The science of miniaturization”, CRC Press, 2002manuals and literature from companies (e.g., AXSUN Technologies, Ca,USA). The choice of materials for fabrication include Nickel (Ni),Nickel-Iron (NiFe), Nickel-Cobalt (Ni—Co) Gold, Copper and Silver. Inthis example, Nickel is selected as the material from which themicro-nanoprojection array template is constructed.

It should be noted here that as an alternative, LIGA could be used tomake individual patches directly (e.g., of gold or silver-which are bothbiocompatible) in large quantities. With current costs of the LIGAtechnology, this is not practical—but with LIGA technology advances,this could be feasible in the future.

Step 2: Construction of a Mask (Female Component)

Because of the discussed current characteristics of LIGA (e.g., cost),LIGA is not used to mass-fabricate micro-nanoprojection array patchesfor direct use with the tissue in large numbers. However, here it can beused as a template for many multi-stage processes. As one example, thetemplate is used for multiple insertions into a soft polymer to producea mask, as shown in FIGS. 30A-30C, performed in the following steps:

FIG. 30A Insertion of the template directly into the soft polymer. Thisis repeated several times to produce many indentations in the polymerwith a given mask.

FIG. 30B Allowing the soft polymer to harden, or “cure” with theaddition of catalysts and or temperature.

FIG. 30C Remove the template to leave behind a mask.

Step 3: Final Construction of the Nanoneedle Array with the Mask

The masks are then be placed in a vacuum chamber and deposited with avacuum deposition/sputtering process. The material to be sputtered is abiocompatible inert material, such as titanium, gold, or silver. In thisprocess, the polymer surface is treated with an air discharge before thechamber is pumped down to a vacuum at 27 degrees C. The titanium, goldor silver film is then deposited. Commercial sputtering machines may beused in this process (such as the VarianVM8 Sputterer).

If a charge is required to produce an anode to enhance the coating ofthe nanoneedle section, then the end extension of this piece can be“opened up” to expose a stronger anode which the positive chargedmetallic ions in solution are attracted to. This technique makes use ofMagnetron Sputtering.

Step 4: Constructional Embodiment Example 3 Method

The mask can then be removed by immersion of a liquid (e.g., alcohols)to dissolve it.

9.3 Constructional Embodiment Example 3

Alternatively, a mask (female) can be directly manufactured usingTwo-Photon Stereo Lithography (2PSL), in which the geometry co-ordinatesshown in FIG. 30 (C) are applied to construct the desired shape with thephotosensitive resin.

Femtosectond two-photon stereo-lithography (2PSL) is described in detailby Miwa et al. (2001, Appl Physics. A 73, 561-566). Zhou et al. (2002,Vol 296, Science), Stellacci et al. (2002, Adv. Mater. 14 (3)) and Haliket al. (2003, Chem Commun 1490-1491). This to approach has demonstratedthe ability to construct complicated 3D shapes with a resolution of <200nm out of materials including photosensitive polymer resins and metalsin conjunction with dyes. Lattice structures of the desired scale havebeen constructed with the technique using resins impregnated with Silver(from Stellacci et al. 2002).

Briefly, the technique works by scanning with a femtosecond laser in aliquid photosensitive resin bath. The two-photon effect ensures thatsolidification occurs only where the energy of the laser is concentratedto a femtolitre volume (typically 200 nm×200 nm×400 nm, x, y, z). Themethod of manufacture is to start at the tip of the nanoneedle (marked Ain FIG. 29) and then work up the needle to the base.

The same approach applies to the rest of the nanoneedles. The process isfully automated with a motorized x-y stage with the laser co-ordinatesdetermined from engineering drawings of the structure. Therefore,thousands of nanoneedles can be constructed in a scaleable process. Thetechnique allows complicated 3D structures to be constructed by thescanning of a pulsed (80 MHz), femtosecond pulse length laserconcentrating light at 400-1000 nm to a femtolitre volume to induce atwo photon excitation of the material and induce solidification.

The nanoneedles may be constructed with a photosensitive polymer/resin,(such as the commercial grade SCR 500) or an alternative impregnatedwith Silver (following Stellacci et al. 2002).

These materials are currently not suited to be used directly in the skindue to insufficient stiffness. Young's Modulus of SCR 500 is 0.49 Gpacompared with

-   -   116 Gpa for Titanium or 77.2 Gpa for Gold.    -   Not medical grade material. At the time of writing,        photosensitive resins such as SCR 500 had not gained approval        for medical grade purposes.

However, the technique is rapidly improving and may be a possiblefabrication method in the future.

9.4 Constructional Embodiment Example 4

In this case nanoneedles are constructed with silicon, with a 200 nm tipfor a length of 5 μm followed by two other parallel sections separatedby tapered sections. This structure may be constructed by Electron BeamLithography and Reactive Ion Etching, a standard technique in to themicroelectronics industry. The manufacturing approach has been appliedby Henry et al. (Journal of Pharmaceutical Sciences 87(8), 1998) and inLebouitz and Pisano et al. (U.S. Pat. No. 5,928,207) in the constructionof microneeedles.

FIG. 31 shows the structure produced with one of these techniques. Inthis case of the nanoneedles, each parallel section may be constructedwith silicon 100 wafers and the tapered sections constructed withsilicon 111 (or other) wafer material—where the angle of the taper is apreferred etching line of the material. The shown shape is made from 5wafers (one wafer per geometry). A gradual taper mold can be constructedfrom the one silicon wafer.

Alternatively, the silicon arrays may be used as masters for microarraymolds.

10. Methods of Treatment

Recall in section 1 the specific target sites of key cells andorganelles are described, followed in section 2 by their location withinmany tissue and in-vitro sites. With the described approach of targetingindividual cells, then several cells and the shape/coating/fabricationconsiderations outlined, the methods of applying thesemicro-nanoprojection arrays to various tissue sites are now discussed.

10.1 Intradermal Application

With the described embodiments for the skin previously described, thepatch is inserted into the skin either by user control or by mechanical,electrical or other controlled means. All these options are possible, asthe magnitude of the forces is low. For example, the insertion force isapproximately calculated to be less than 1 N (based on 7000 probes, 1 μmin diameter piercing tissue with a yield of 20 MPa).

Similarly, the time of insertion, residence and removal from the tissuecan be controlled by user and/or the described mechanical/electricalmeans.

One embodiment of a simple application system for the patch is shown inFIGS. 32A and 32B.

In this example, the application system 3300 is formed from a structurein the form of a housing or body 3310. The body 3310 defines a cavity3320 having an opening 3330. In use, a patch 100, having a number ofprojections 110 provided on a base 120, is moveably mounted within thecavity 3320. This may be achieved in any one of a number of manners, tobut typically involves having the patch 100 suitable sized to allowmovement along the cavity towards and away from the opening 3330.

The patch 100 is mounted to an actuator, so that in use the patch may bemoved from a retracted position shown in FIG. 32A, to an extendedposition shown in 32B. The actuator may be of any suitable form, but inone example, includes a spring 3340 and a releasing means 3350.

In the retracted position, the spring 3340 is biased against the patch100, thereby urging the patch 100 towards the opening 3330, with thepatch 100 being retained in the retracted position by the releasingmember 3350. When the releasing means is activated, for example, byhaving an operator release the wire, the spring 3340 urges the patch 100towards the opening 3330, so that the projections 110 extendtherethrough.

It will be appreciated from this that if the structure 3310 ispositioned within the opening 3330 adjacent a subject's skin 3360, thenoperation of the releasing means 3350 causes the patch 100 to be pressedagainst the subject's skin 3360, so that the projections 110 enter theviable epidermis as described above.

In the retracted position (FIG. 32 (a)) the micro-nanoprojections patchis recessed and thus protected, preventing accidental administration.Other packaging/devices may also be used for these purposes, for examplea protective cap to be removed before administration.

In this example, the patch is attached to a compressed spring that isheld in place by a tensioned string. It will be appreciated that thereleasing means typically is arranged so that the patch 100 is retainedin the retracted position until the releasing means is activated. Thus,the patch 100 may be retained in position by a clip that is releasedupon activation of the releasing means. This allows the operator toposition the device against the subject's skin 3360 without having toretain the patch in the retracted position. When located on the tissue,the string, is released by a means (e.g., pressing a button, not shownin FIG. 32) and the patch is released to enter the tissue surface (FIG.32(b)).

10.2 Mucosal Application

To effectively target mucosal sites (e.g., mouth, nasal, rectal,vaginal), the described patch arrangements may be fitted to anapplicator designed to safely transport the patch into the mouth and toaccurately locate on to the mucosal site. This could be done by usingthe described intradermal patch in the mouth.

Alternatively, FIG. 33 shows one example of and applicator for mucosaldelivery for the buccal mucosa (mouth). In this example, the patcharrangement from FIG. 32 in the form of the application system 3300 iscoupled to an arm 3400, allowing the application system 3300 to bepositioned in otherwise hard to reach places. The spring arrangementallows the patch to be applied, but other mechanisms equally could beused. The applicator could have a button at the distal end of the arm3400 from the patch to allow the operator to release the patch onto themucosal site.

Other features may include a knurled surface towards the distal end toaid user grip.

10.3 Lower Airway/Lung Application

FIG. 34 shows a schematic of the physiology of the airways in a human.To apply the micro-nanoprojections to targeting cells in the tracheal orlung lining, an applicator that can flexibly and compactly reach thesesites, target the sites, and then retract, is required.

As one example, FIGS. 35A-35C show a deployable structure embodiment fortargeting these sites.

In this example, the apparatus includes an application system 3600mounted to a flexible structure, such as an arm, allowing theapplication system 3600 to be inserted into a passageway 3620, such asan airway, of the subject. To allow the application system 3600 to beguided to the correct location, the flexible structure may be in theform of a manipulable fiberscope, or the like.

The application system includes a structure such as a body 3630, havinga number of arms 3640 movably mounted thereto, to allow the arms to movebetween a retracted position shown in FIG. 35B and an extended positionshown in FIG. 35C. In use, the arms 3640 are typically biased towardsthe extended position, and are retained in the retracted position, usinga releasing mechanism, such as a clip or the like. It will beappreciated that this may be similar to the releasing means describedabove with respect to FIG. 34.

In any event, patches 100 are mounted to the arms, so that when the armsare released, the patches are urged against a surface 3621 of thepassageway 3620, allowing the projections 110 provided on each patch topenetrate the surface 3621, and deliver material or stimulus toprospective targets.

In the example shown, the eight arms 3640 are provided in two sets offour, with the four arms in each set being circumferentially spacedaround the body 3630, as shown in FIG. 35C.

The method of operation is as follows. The flexible structure (FIG. 35A)is guided through the throat to the site of interest. This device may befitted with imaging/illumination systems to help guidance. FIG. 35Bshows the system in location in a “retracted” position. The arms fittedwith the patches are held in place against a spring load with pivotpoints. Then, by mechanical actuation, the arms are released (FIG. 35C),with the springs providing the force for location on the tissue.

The device does not need to be exactly centralized as the arms areself-locating. By tension with wires, the arms are retracted, and thedevice is removed through the throat.

In another embodiment, the arms are replaced by an inflatable structure(a “sock”) fully coated by the micro-nanoprojections on the outsidesurface. When deflated, this sock would be held in a structure whichwould look similar to FIG. 35A on the outside. When in place, the sockwould be inflated via a pressurized gas, locating on the tissue wall.This approach would be particularly useful where large surfaces needtargeting, such as the lung. At the required time, the pressure isreleased with a valving arrangement and the sock collapses back intoflexible targeting system and the device is removed.

10.4 Other Internal Tissues

Other internal organs or tissues (e.g., liver, kidney, heart) are not asreadily accessed as those described above. Here, more invasive means arerequired to expose the site of interest before targeting. One example isa more compact “catheter” version of the described lower airway/lungtargeting devices, reaching the site via keyhole routes.

Another embodiment is surgery to fully open the site before applicationwith the patch.

10.5 In-Vitro Sites

The micro-nanoprojections may be fitted to patches for in-vitrotargeting, allowing a high-throughput targeting of cells. In oneexample, larger patches with thousands and perhaps millions ofprojections could be mechanically lowered onto cell monolayers, and thenremoved, similar to a mechanical “press” arrangement. This could, forinstance, allow a mass transfection of cells, in-vitro.

As another variation, cells are dynamically moving in a shallow fluidstream. As these cells move, they pass below large plates withthousands, and perhaps millions of micro-nanoprojections. These platespressing into this cell layer repeatably, in a synchronized manner sothat each pressing cycle targets the appropriate batch of cells.

1. A device for delivery of material to targets within a body to producea desired response, the device including a number of projections forpenetrating a body surface, and wherein: a) the projections include atargeting section for delivering the material to the targets to therebycause the desired response; b) the number of projections is at least500; c) a spacing between projections is between 1 and 1000 μm; and d)at least part of the at least some of the projections are coated with anon-liquid material. 2.-5. (canceled)
 6. A device according to claim 1,wherein the number of projections is 1000 to
 10000. 7. (canceled) 8.(canceled)
 9. A device according to claim 1, wherein the spacing betweenat least some of the projections is selected to avoid multipleprojections targeting a single target of interest.
 10. A deviceaccording to claim 1, wherein the spacing between at least some of theprojections is selected to be greater than a diameter of the targets ofinterest.
 11. A device according to claim 1, wherein the spacing betweenat least some of the projections is selected to be approximately equalto the spacing between the targets of interest.
 12. A device accordingto claim 1, wherein the spacing between the projections is 10 μm≤S≤200μm. 13.-15. (canceled)
 16. The device of claim 1 wherein the projectionsfurther comprise a support section. 17.-21. (canceled)
 22. A deviceaccording to claim 16, wherein a length for the support section is <1000μm.
 23. A device according to claim 1, wherein a length of thetargeting, section is greater than the target dimension.
 24. A deviceaccording to claim 1, wherein at least some of the projections havedifferent dimensions.
 25. A device according to claim 1, wherein theprojections are solid.
 26. A device according to claim 1, wherein theprojections are non-porous and non-hollow.
 27. A device according toclaim 1, wherein at least part of at least some of the projections arecoated with a bioactive material. 28.-30. (canceled)
 31. A deviceaccording to claim 1, wherein the device includes at least some uncoatedprojections to thereby stimulate or perturb the targets in use. 32.(canceled)
 33. A method of selecting constructional features for adevice for delivery of material or stimulus to targets within a body toproduce a desired response, the targets being at least one of cells ofinterest, cell organelles of interest and cell nuclei of interest, thedevice including a number of projections for penetrating a body surface,and wherein the method includes: a) selecting the number of projectionsto produce a desired response, the number being at least 500; and, b)selecting a spacing between projections at least partially based on anarrangement of the targets within the body.
 34. A method of fabricatinga device for delivery of material or stimulus to targets within a bodyto produce a desired response, the targets being at least one of cellsof interest, cell organelles of interest and cell nuclei of interest,the device including a number of projections for penetrating a bodysurface, and wherein the method includes: a) selecting the number ofprojections to produce a desired response, the number being at least500; b) selecting a spacing between projections at least partially basedon an arrangement of the targets within the body; and, c) fabricatingthe device using the selected number of projections, and the selectedspacing.
 35. A method of treating a subject the method including using adevice for delivery of material or stimulus to targets within thesubject's body to produce a desired response, the targets being at leastone of cells of interest, cell organelles of interest and cell nuclei ofinterest, the device including a number of projections for penetrating abody surface, and wherein: a) the number of projections is selected toproduce a desired response, the number being at least 500; and, b) aspacing between projections is at least partially determined based on anarrangement of the targets within the body. 36.-45. (canceled)
 46. Thedevice according to claim 1 wherein at least part of at least some ofthe projections are coated with two or more non-liquid materials on thesame projection.
 47. The device according to claim 1 wherein theprojections are made of one or more polymer(s).
 48. The device accordingto claim 1 wherein the projections are made of one or morephotosensitive polymer resin(s).
 49. The device according to claim 1wherein the projections comprise a first projection and a secondprojection wherein the first projection is coated with a first coatingmaterial and the second projection is coated with a second coatingmaterial and wherein the first coating material and the second coatingmaterial are different materials.
 50. The device according to claim 1wherein the projections are arrayed with a density of 10 to 100projections per mm².
 51. A device for delivery of a vaccine beneath theskin of a patient to produce an immunological response, the deviceincluding a number of projections for penetrating the skin and wherein:a) the projections include a targeting section for delivering thematerial to the targets to thereby cause the immunological response; b)the number of projections is from 1000 to 10,000; c) a spacing betweenprojections between 10 to 200 μm; and d) at least part of the at leastsome of the projections are coated with a non-liquid vaccine material.